Resistant Starch with Cooking Properties Similar to Untreated Starch

ABSTRACT

A method has been discovered to produce a resistant starch product that retains the same cooking quality as found in untreated rice starch or flour, but has a higher percentage of starch resistant to α-amylase digestion. This method uses a debranching enzyme, e.g., pullulanase, to digest the starch, but does not require pre-treating the starch source prior to enzymatic treatment. This method produced resistant starch from low amylose starches, rice starch (24%) and rice flour (20%). Surprisingly the resistant starch product formed by this method retained the pasting characteristics of the untreated flour or starch, and was heat stable. This method may also be used to produce resistant starch from other botanical sources, e.g., corn, wheat, potato, oat, barley, tapioca, sago, and arrowroot. Resistant starch produced by this method has a variety of uses in food products.

This is a divisional of copending application Ser. No. 10/936,116, filedSep. 7, 2003, which claims the benefit of the filing date of provisionalapplication 60/501,121, filed Sep. 8, 2003 under 35 U.S.C. § 119(e).

This invention pertains to a resistant starch produced from a nativestarch, e.g., rice starch or rice flour, that retains the pastingcharacteristics of the native starch, and to a new method to producethis resistant starch.

The beneficial effects of resistant starch are well known. However, mostmethods of producing resistant starch begin with a starch that is atleast 40% amylose, usually from corn. These methods usually do not workwell with rice starches since even high amylose starch from rice is onlyabout 27% amylose. See, U.S. Pat. No. 6,303,174. Other sources of starchinclude wheat, oat, barley, tapioca, sago, cassava, potato, andarrowroot.

One advantage to rice is that people who are allergic to wheat often donot have problems with rice. Use of rice as a food ingredient accountsfor 22% of domestic rice sales. This use has increased by 3.7%, due tothe rising popularity and availability of snacks, frozen dinners, ricepudding, package mixes and candy. Pet food products are alsoincorporating rice as an ingredient. Even though rice contains only 7 to8% protein, the protein quality is high and also is high in theessential amino acid, lysine. In contrast, most other grains aredeficient in lysine. Rice is approximately 87% carbohydrates, but ricestarch contains less amylose than other high amylose grains, e.g.,potato and corn. Rice starch consists primarily of amylose andamylopectin.

Starch

Starch is primarily a mixture of two polymers of glucose residues:amylose and amylopectin. In untreated starch, the two polymers arepacked into discrete particles (granules). Particle size ranges from2-100 μm. At 80° C. (175° F.), unmodified starch granules form a pastewith very high viscosity, as the starch granules swell and aredisrupted. See O. R. Fennema, Ed., 3^(rd) ed, Food Chemistry, Ch. 4,“Carbohydrates,” Marcel Dekker, Inc. New York, pp. 167-168, 174, 195,196 (1996). When the starch is cooled, retrogradation occurs as amyloserecrystallizes. See S. Rashmi et al, “Effect of processing onnutritionally important starch fractions in rice varieties,”International Journal of Food Sciences and Nutrition, vol. 54, pp. 27-36(2003).

Starch is insoluble in cold water and can imbibe water reversibly. Whenheated in water, starch can undergo gelatinization as starch granulesswell. Gelatinization can be irreversible if the starch granules are sodisrupted to cause excess starch granule swelling and loss ofbirefringence and crystallinity (Fennema, 1996). Gelatinization is aprocess that normally occurs over a temperature range of approximately10 to 15° C. The gelatinization temperature range for waxy, normal ricestarch with about 50% water is in the range of 61-93° C. If this ricestarch contains about 20% amylose, it gelatinizes between 60 and 78° C.See D. J. A. Jenkins et al., “Low glycemic index: Lente carbohydratesand physiological effects of altered food frequency,” Am. J. Clin.Nutr., vol. 59, p: 706S (1994); and A. W. Thorburn et al., “Slowlydigested and absorbed carbohydrate in traditional bushfoods: Aprotective factor against diabetes?” Am. J. Clin. Nutr., vol. 45(1),pp.: 98-106 (1987). The degree of gelatinization is affected by a numberof factors, such as temperature, starch:water ratio, granule type,measurement technique, granule heterogeneity within the starch sample,and actual botanical source of starch. See Fennema, 1996; A. C. Eliassonet al., “Ch 10. Starch: Physicochemical and Functional Aspects,” In,Carbohydrates in Food, pp. P441-443 (1996); and Z. Ming et al., “Sourcesof variation for starch gelatinization, pasting, and gelation propertiesin wheat,” Cereal Chem., vol. 74, pp. 63-71 (1997). One method tomeasure gelatinization temperature is by differential scanningcalorimetry (DSC). See Eliasson et al., 1996; Fennema, 1996; C. Sievertet al., “Enzyme-resistant starch. II. Differential scanning calorimetrystudies on heat-treated starches and enzyme-resistant starch residues,”Cereal Chem., vol. 68, pp. 86-91 (1990); D. Sievert et al.,“Enzyme-resistant starch. II. Differential Scanning Calorimetry Studieson Heat-Treated Starches and Enzyme-Resistant Starch Residues,” CerealChem., vol. 67(3), pp. 217-221 (1990).

Retrogradation of starch occurs as dissolved starch becomes less solubleand more ordered in a crystalline state. (Fennema, 1996) The rate ofretrogradation is dependent on the molecular ratio of amylose toamylopectin, the structure of the amylose and amylopectin molecules(source of starch), temperature, starch concentration, andconcentrations of other ingredients such as surfactants and salts. Ingeneral, retrogradation occurs to a greater extent with higher linearamylose content. Shelf-life and quality of food products can beshortened or decreased due to retrogradation, e.g., bread staling, lossof viscosity and precipitation in soups and sauces. (Fennema, 1996)

When starch is heated past gelatinization in excess water so that thestarch granules swell and become totally disrupted, a viscous mass(paste) is formed, a process called pasting. The rapid visco amylograph(RVA) measures the pasting characteristics of starch in rapid viscounits (RVU). RVA is unable to measure gelatinization onset because thestarch granules are gelatinized before viscosity begins to increase andbe detected. See X. M. Liang et al., “Pasting Property Differences ofCommercial and Isolated Rice Starch with Added Lipids andβ-Cyclodextrin,” Cereal Chem., vol. 79, pp. 812-818 (2002). The pastingcharacteristics determine the cooking properties of the starch and areuseful in determining the use in food products. RVA has been used toinvestigate the pasting effects of lipids and amino acids on rice starchand flour. See Liang et al., 2002; and X. M. Liang et al., “Pasting andCrystalline Property Differences of Commercial and Isolated Rice Starchand Added Amino Acids,” Journal of Food Science, vol. 68, pp. 832-838(2003).

There are three categories of starches based on digestion properties:readily digestible (RDS), slowly digestible (SDS), and resistant starch(RS). Resistant starch is defined as starch that has the ability tosurvive prolonged incubation with α-amylase and thus passes undigestedinto the large intestine in humans. See H. N. Englyst et al.,“Classification and measurement of nutritionally important starchfractions,” European Journal of Clinical Nutrition, vol. 46 (Suppl. 2),pp. S33-S50 (1992); and C. S. Berry, “Resistant starch: Formation andmeasurement of starch that survives exhaustive digestion with amylolyticenzymes during the determination of dietary fiber,” J. Cereal Sci., vol.4, pp. 301-314 (1986). Resistant starch can be further divided into foursubgroups: RS1 which is physically inaccessible starch, RS2 which isfood that is often eaten raw or cooked with very little water so thatthe granular structure is intact, and RS3 as retrograded amylose.(Englyst et al., 1992). RS3 has been characterized as native starchgranules that have been gelatinized and retrograded afterwards.(Eerlingen, 1994) As the amylose content in the starch increases, thedegree of retrogradation increases. RS3 formation is highly dependent onamylose content, temperature, prior gelatinization, presence of lipids,proteins and sugars, and source of starch. (Fennema, 1996). High amylosestarch was also found to be more resistant to digestion than amylopectindue to its compact linear structure. (Rashmi et al., 2003) A fourth typeof RS has been developed by treating starch with chemicals. (Eerlingen,1994)

Amylose is a linear chain of (1→4)-linked α-D-glucopyranosyl units withsome α-D-(1→6) side branches. Alpha-D-(1→6) branches may occur once inevery 180-320 units, or in about 0.3-0.5% of the linkages. The molecularweight of amylose is approximately 10⁶ Daltons. Most starches containabout 25% amylose, but some can have up to 70% amylase (e.g., Hi-Maize™developed by Penford Ingredients; Denver, Colo.). Amylose content isconsidered the main parameter in starch that determines cooking andeating quality in rice. Amylose content in rice ranges from 18-35% andvaries with geographic regions. (TropRice, 2003). Milled rice isclassified based on amylose content: waxy (1-2%); non-waxy (>2%); verylow (2-9%); intermediate (20-25%); and high (25-33%). Rice grown inMissouri had some 3-18% higher amylose content and a higher proportionof short linear chain amylopectin than the same rice varieties grown inArkansas or Texas. See A. Aboubacar et al., “The effects of growthlocation on US rice starch structure and functionality,” Whistler Centerfor Carbohydrate Research and Dept. of Food Science, Purdue University.West Lafayette, Ind. 47907-1160 (2002). RVA (rapid visco amylograph)analysis indicated that rice grown in Missouri had lower peak (1-26%)and breakdown (3-43%) viscosities than both the Arkansas- andTexas-grown rice. Rice varieties with similar amylose content have beenreported to have different starch digestibility. See L. N. Panlasigui etal, “Rice varieties with similar amylose content differ in starchdigestibility and glycemic response in humans,” Am. J. Clin. Nutr., vol.54, pp. 871-7 (1991).

Amylopectin is a highly branched polymer with a molecular weight from10⁷ to 5×10⁸, making it one of the largest polymers in nature.Amylopectin is about 75% of most starches. It consists of both (1→4) and(1→6) α-D-glucopyranosyl units. Starches made of 100% amylopectin arecalled waxy starches, even though there is no wax present. The term“waxy” is used to describe the vitreous or waxy surface when a kernel iscut. Amylopectin is found in the highest proportion in medium, short,and waxy rice, and causes these types of rice to be softer and have agreater tendency to cling. Texture of cooked rice depends on the ratioof amylopectin to amylose.

Digestion of Starch

The hydrolytic enzymes used to digest starches are classified into twotypes, endo- and exo-enzymes, which digest starch into different endproducts. For example, amyloglucosidase (glucoamylase), an exo-enzyme,is used commercially to convert starch into glucose. See R. Manelius,“Enzymatic and Acidic Hydrolysis of Native and Modified StarchGranules,” Acta Academiae Aboensis, Ser. B., vol. 60(2), pp. 20-21(2000). Using this enzyme and pre-gelatinized starch, the starch iscompletely converted to glucose. Glucoamylase cleaves successive α(1,4)and α(1,6)-D-glucosidic linkages from the non-reducing end to produceglucose.

Alpha-Amylase is an endo-enzyme that cleaves α(1,4)-D-glucosidiclinkages in starch. The end products after α-amylase digestion ofamylopectin are glucose, maltose, maltotriose, and branched α-limitdextins (pentasaccharides). See D. French et al., “The structuralanalysis and enzymic synthesis of a pentasaccharide alpha-limit dextrinformed from amylopectin by Bacillus subtilis alpha-amylase,” Carbohydr.Res., vol. 22, pp. 123-134 (1972). On the other hand, pullulanase is adebranching endo-enzyme that cleaves the α(1,6) linkages, especiallywhen separated by at least 2 glucose residues joined by α(1,4) linkages.(Manelius, 2000). Other debranching enzymes, generally termedendo-alpha-1,6-glucanohydrolases, are known such as isoamylase or anyother endo-enzyme that exhibits selectivity in cleaving the 1,6-linkagesof the starch molecule, leaving the 1,4-linkages substantially intact.

Resistant Starch Formation

Formation of resistant starch type III (RS3) depends on many factors,e.g., pH, temperature, incubation time, storage time, number of heatingand cooling cycles, type of starch, and water content. Amylose contentand amount of water has been directly correlated to resistant starchyield. See C. Sievert et al., “Enzyme-resistant starch. I.Characterization and evaluation by enzymatic, thermoanalyical andmicroscropic methods,” Cereal Chem., vol. 66, pp. 342-347 (1989).

Resistant starch can be formed through retrogradation. Retrogradation isthe precipitation of starch molecules in cooled pastes and gels thatcontain mainly amylose. The hydrogen bonds within hydrated starchinteract, resulting in physical-chemical changes without the creation ofpermanent chemical bonds. (Berry, 1986). Amylopectin retrogrades veryslowly. High amylose starches have a greater retrogradation.Additionally, high amylose starch is more resistant to digestion thanamylopectin due to its compact linear structure (Rashmi et al, 2003).Factors that determine rate of retrogradation are the molecular ratio ofamylose to amylopectin, the structure of the amylose and amylopectin,temperature, starch concentration, and concentrations of otheringredients, e.g., sugars. See Fennema, 1996; P. L. Russell et al.,“Characterization of resistant starch from wheat and maize,” J. CerealSci., vol. 9, pp. 1-15 (1989); and T. Sasaki et al., “Effect of AmyloseContent on Gelatinization, Retrogradation, and Pasting Properties ofStarches from Waxy and Nonwaxy Wheat and Their F1 Seeds,” Cereal Chem.,vol. 77, pp. 58-63 (2000).

When gelatinization occurs in the presence of excess water, resistantstarch (RS3) formation is greatly enhanced by retrogradation.Significantly higher levels of RS have been found in cooked pasta thanbread. Repeated cycling of autoclaving and cooling, up to 20 cycles,increased RS formation from 20 to over 40%. By raising the autoclavetemperature from 121 to 134° C., a decrease in RS yield was seen(Sievert et al, 1989).

Amylose content in starch affects RS yield since RS is retrogradedamylose. Amylose will also bind with lipids, proteins and othercompounds. The formation of amylose-lipid complexes is reported tocompete with and be favored over amylose retrogradation, thus decreasingthe RS yield. See R. C. Eerlingen et al., “Enzyme-resistant starch. IV.Effect of endogenous lipids and added sodium dodecyl sulfate onformation of resistant starch,” Cereal Chem., vol. 71(2), pp. 170-177(1994); and L. Slade et al., “Starch and sugars as partially-crystallinewater-compatible polymer systems,” Cereal Food World, vol. 32(9), p. 680(1987). Enzymes, such as α-amylase, amyloglucosidase, and pullulanase,have been used to treat waxy and normal maize starches to produce RSafter gelatinization. (Berry (1986) Treating amylomaize and amylopectinstarches with pullulanase followed by heat yielded higher RS levels thanheating alone. Using both heating and pullulanase, RS yields increasedin amylomaize and amylopectin starches from 0.3 to 32.4% and from 4.2 to41.8%, respectively.

Potential Benefits of Resistant Starch

Resistant starch is beneficial in part because as undigestible dietaryfiber, it provides bulk to aid in gut peristalsis and thus decrease thetransit time of food/waste in the intestine. By consuming 35 gfiber/day, chances of constipation were lower by 60% and heartburn by30%. Dietary fiber has also been found to help lower cholesterol. See P.Yue et al., “Functionality of resistant starch in food applications,”Food Australia, vol. 50, pp. 615ff, as reprinted by National Starch &Chemical Company (1998). Hypercholesterolemic patients that consume upto 50 g dietary fiber/day are benefited by maintaining a normal level ofserum cholesterol. Dietary fiber can also lower postprandial serumglucose levels and insulin response by slowing starch digestion.(Fennema, 1996) As a way to increase fiber in the diet, resistant starchcan help prevent colon cancer, lower the risk of heart disease, andinfluence metabolic and inflammatory bowel diseases, such as diabetesand diverticulitis. RS is also a prebiotic because it produces butyrateand other short-chain fatty acids when fermented in the large intestine.

U.S. Pat. No. 4,971,723 discloses a method to produce a partiallydebranched starch by treating a pre-gelatinized starch with adebranching enzyme, an endo-alpha-1,6-D-glucanohydrolase.

U.S. Pat. No. 5,051,271 discloses a method to produce a food-grade,water insoluble material with water soluble crystalline microparticlesby causing the initial starch to undergo retrogradation using a heatingand cooling cycle, followed by enzymatic hydrolysis.

U.S. Pat. Nos. 5,281,276 and 5,409,542 disclose a product and a methodto increase the yield of resistant starch from a high amylose starch (atleast 40%) by initially gelatinizing the starch by heating, followed byincubating the starch with a debranching enzyme for 24 to 48 hours.

U.S. Pat. No. 5,395,640 discloses a method to prepare reduced fat foodsby adding a debranched amylopectin starch that is made by gelatinizingthe starch, followed by enzymatic debranching.

U.S. Pat. No. 5,480,669 discloses a method to improve the texture offood products with a high fiber content by incorporating resistantstarch into the dough, where the resistant starch was made frominitially gelatinizing the starch and then debranching enzymatically.

U.S. Pat. Nos. 5,593,503 and 5,902,410 disclose a method to prepare aresistant granular starch from a starch source with at least 40% amyloseby heating the starch using a combination of moisture and temperatureconditions.

U.S. Pat. No. 5,849,090 discloses a method to make granular resistantstarch by heating the starch initially to a temperature from about 60°C. to about 120° C. to swell the starch granules, debranching theswollen starch, and then treating the starch product to retrograde theamylose.

U.S. Pat. No. 5,962,047 discloses a method to produce resistant starchby treating a hydrated starch source, which is optionally debranched, tocause retrogradation, and then to cause enzymatic or chemicalhydrolysis.

U.S. Pat. No. 6,043,229 discloses a method to produce resistant starchfrom a partially degraded starch product (prepared by enzymatic or acidhydrolysis, e.g., potato maltodextrin) using enzymatic debranching withan optional retrogradation step.

U.S. Pat. No. 6,468,355 discloses a method to produce a heat stablestarch product with up to 60% resistant starch by partially hydrolyzingthe starch with acid, followed by heating the partially hydrolyzedstarch.

There is a need for new methods to increase the resistant starch yieldfrom low amylose starches, and to form resistant starch with bettercooking properties.

We have discovered a method to produce a resistant starch product thatretains the same cooking quality as found in untreated rice starch orflour, but has a higher percentage of starch resistant to α-amylasedigestion. This method uses a debranching enzyme, e.g., pullulanase, todigest the starch, but does not require pre-treating the starch sourceprior to enzymatic treatment. The starch source is neither hydrolyzednor gelatinized before adding the enzyme. The incubation temperature ofthe starch and enzyme stays below 60° C. This method produced resistantstarch from low amylose starches, rice starch (24%) and rice flour(20%). Surprisingly the resistant starch product formed by this methodretained the pasting characteristics of the untreated flour or starch,and was heat stable. The highest yield of resistant starch using thisnew method was produced from rice starch, up to twelve-fold higher thanthat found in the native starch. Our best results to date of productionof resistant starch with the desired pasting characteristics and heatstability were obtained by incubating the untreated starch withpullulanase at a temperature between about 40° C. and about 60° C.,preferably about 55° C., for an incubation period from about 2 hr toabout 16 hr, preferably from 2 hr to 4 hr. This method may also be usedto produce resistant starch from other botanical sources, e.g., corn,wheat, potato, oat, barley, tapioca, sago, cassava, and arrowroot.Resistant starch produced by this method has a variety of uses in foodproducts.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the results of rapid visco amylograph analysis, anindication of pasting characteristics, of untreated rice flour,untreated rice starch, and a commercial resistant starch, CrystaLean®.

FIG. 2 illustrates the results of rapid visco amylograph analysis ofnon-gelatinized rice flour that was not stored before being incubatedwith the enzyme pullulanase for either 2 hr (NGNS2hr), 4 hr (NGNS4hr),or 16 hr (NGNS16hr).

FIG. 3 illustrates the results of rapid visco amylograph analysis ofgelatinized rice flour that was not stored before being incubated withpullulanase for either 2 hr (GNS2hr), 4 hr (GNS4hr), or 16 hr (GNS16hr).

FIG. 4 illustrates the results of rapid visco amylograph analysis ofgelatinized rice flour that was stored overnight before being incubatedwith pullulanase for either 2 hr (GS2hr), 4 hr (GS4hr), or 16 hr(GS16hr).

FIG. 5 illustrates the results of rapid visco amylograph analysis ofnon-gelatinized rice starch that was not stored before being incubatedwith the enzyme pullulanase for either 2 hr (NGNS2hr), 4 hr (NGNS4hr),or 16 hr (NGNS16hr).

FIG. 6 illustrates the results of rapid visco amylograph analysis ofgelatinized rice starch that was not stored before being incubated withpullulanase for either 2 hr (GNS2hr), 4 hr (GNS4hr), or 16 hr (GNS16hr).

FIG. 7 illustrates the results of rapid visco amylograph analysis ofgelatinized rice starch that was stored overnight before being incubatedwith pullulanase for either 2 hr (GS2hr), 4 hr (GS4hr), or 16 hr(GS16hr).

FIG. 8 illustrates the results of differential scanning calorimetry onuntreated rice flour, untreated rice starch, and a commercial resistantstarch, CrystaLean®.

FIG. 9 illustrates the results of differential scanning calorimetry ofnon-gelatinized rice flour that was not stored before being incubatedwith the enzyme pullulanase for either 2 hr (NGNS2hr), 4 hr (NGNS4hr),or 16 hr (NGNS16hr).

FIG. 10 illustrates the results of differential scanning calorimetry ofgelatinized rice flour that was not stored before being incubated withpullulanase for either 2 hr (GNS2hr), 4 hr (GNS4hr), or 16 hr (GNS16hr).

FIG. 11 illustrates the results of differential scanning calorimetry ofgelatinized rice flour that was stored overnight before being incubatedwith pullulanase for either 2 hr (GS2hr), 4 hr (GS4hr), or 16 hr(GS16hr).

FIG. 12 illustrates the results of differential scanning calorimetry ofnon-gelatinized rice starch that was not stored before being incubatedwith the enzyme pullulanase for either 2 hr (NGNS2hr), 4 hr (NGNS4hr),or 16 hr (NGNS16hr).

PRODUCTION OF RESISTANT STARCH FROM RICE STARCH AND RICE FLOUR

The starches used in preparing resistant starch of this invention may bederived from any source containing amylose, for example, from corn,potato, barley, sweet potato, wheat, rice, sago, tapioca, cassava, andsorghum. The method of this invention will work on starches with bothhigh amylose content and low amylose content (less than 30%).

Unlike other methods of producing a product high in resistant starch,the starch is not pre-gelatinized or pre-hydrolyzed prior to treatmentwith a debranching enzyme. The debranching enzyme, anendo-alpha-1,6-glucanohydrolase, is added to the native starch, and themixture is then heated to the optimum temperature for the enzyme. Forexample, if the enzyme pullulanase is used, the mixture is heated to atemperature between about 40° C. to less than 60° C., more preferablyabout 55° C. Another debranching enzyme may be used, e.g., isoamylase.See U.S. Pat. No. 5,409,542. Optimum concentrations of the enzyme andsubstrate are governed by the level of enzyme activity, which will varydepending on the enzyme source and concentration. The starch and enzymeincubation in this invention is only about 2 hours to 16 hours,preferably 2 hours to 4 hours, and most preferably about 4 hours. Thisis in stark contrast to those methods that allow the debranching tocontinue for 24 to 48 hours. It is believed that the above method can beused to produce a resistant starch product from starch from any source,including wheat, potato, oat, rice, barley, sorghum, corn, arrowroot,cassava, and sago.

Example 1

Methods for Formation of Resistant Rice Starch Using DifferentTreatments and Enzymes

Preparation of Rice Samples

Rice flour was obtained from Riviana Foods Inc. (Abbeville, La.) inbulk, while rice starch was purchased from Sigma Chemical Co. (S7260 inkilogram quantities; St. Louis, Mo.). CrystaLean®, acommercially-available resistant starch from corn by Opta FoodIngredients (Bedford, Mass.), was used as a control.

To prepare the samples of starch, one hundred grams of rice starch orrice flour were placed in 2-L Erlenmeyer flasks, and 1400 g of distilledwater added. For the “Gelatinized samples” (G), the mixture was stirredand heated on a hot plate to approximately 95° C. The gelatinizedsamples were divided into two subgroups, one without storage (GNS) andone with 24-hr refrigeration at 3° C. (GS). For the “Non-gelatinizedsample” (NG), the mixture was heated only to the recommended temperaturefor optimal performance of the enzyme to be tested, and enzyme addedimmediately without any storage time (NGNS). Samples were prepared induplicate for each treatment.

Enzymatic Treatments and Initial Analyses

Three enzyme-starch incubation periods were tested, 2-, 4-, and 16-hr.Two enzymes were tested, pullulanase (Promozyme™ 400L, Sigma P2986;pullulanase from Bacillus acidopullulyticus; minimum 400 units/ml; SigmaChemical Co.) and α-amylase (Termamyl® 120L, Type L, 120 knu/g; NovoNordisk Biochem, Franklinton, N.C.). Additionally, a combination ofpullulanase-α-amylase was tested. The gelatinized samples were cooled toa temperature that was optimal for each enzyme: 55° C. for pullulanaseand 75° C. for α-amylase treatments. For the combination ofpullulanase-α-amylase, the sample was cooled to 60° C. Thegelatinized/stored samples (GS) were stored overnight in therefrigerator prior to adding enzymes. The NG samples were heated to theoptimal temperatures as given above. Ten-ml of each enzyme to be testedwas added to each sample; i.e., 10 ml pullulanase to a sample treatedonly with pullulanase, 10 ml α-amylase to a sample treated only withα-amylase, and both 10 ml pullulanase and 10 ml α-amylase to a sampletreated with the combination. After the incubation period, the sampleswere centrifuged (Model RC-5C from Sorvall Instruments, DuPont) at 8500rpm for 20 min at 4° C. The residue was collected and frozen at −20° C.,then placed in a freeze-dryer sublimator (20 SRC-X; Virtis Co, Inc.,Gardiner, N.Y.). After freeze-drying, the sample was weighed, thefreeze-dried sample weight (FDSWt). The samples were then milled with aCyclone Sample Mill (Udy Corporation, Fort Collins, Colo.). Moistureremaining in the samples was measured by weighing samples before andafter drying in a Mettler LP16 Infrared Dryer (Mettler-ToledoIncorporation, Hightstown, N.J.).

Resistant Starch Analysis

The total dietary fiber (TDF) content was determined by use of acommercial total dietary fiber kit (Sigma, TDF-100A, St. Louis, Mo.). Inthe TDF analysis, α-amylase and amyloglucosidase were used to digest anydigestible carbohydrate present in the samples. Resistant starch yieldwas then determined by the glucose oxidase assay as described by B. V.McCleary et al., “Measurement of Resistant Starch. Food Composition andAdditives,” J. AOAC International, vol. 85, pp. 665-675 (2002). Theconcentration of resistant starch was determined by digesting the samplewith amyloglucosidase to form free glucose, and then detecting theabsorbance of free glucose in a spectrophotometer. Resistant starchyield (RS Yield) was calculated based on the weight of enzyme-treatedsamples (freeze-dried sample weight, FDSWt), taking into considerationthe moisture content of the samples. True yield (TY) represented thetrue resistant starch yield based on the weight of the originaluntreated rice starch or flour (100 g, including moisture).

In the TDF assay, 200 mg of enzyme-treated, freeze-dried sample wasadded to a 125 ml Erlenmeyer flask. Ten ml of phosphate buffer, pH 6.0,and α-amylase (0.02 ml) (68,300 units/ml) were added, and the samplemixed. The flask was covered with aluminum foil and placed in a boilingwater bath. The sample was then agitated gently at 5-min intervals, andincubated for 15 min after the temperature of the mixture reached 95° C.The solution was then cooled to room temperature, and pH was adjusted tobe within the range 4.0-4.6 by adding 0.375 N HCl. After an appropriatepH was obtained, amyloglucosidase (0.02 ml) (10,863 units/ml; Sigma, A9913) was added. The flask was covered with aluminum foil, placed in a60° C. agitator-incubator, and incubated for 30 min after thetemperature of the sample reached 60° C. Four volumes (10 ml each) 200proof ethyl alcohol was added to the solution to precipitate the starch,and the flask was left overnight at room temperature to allow completeprecipitation. The next day, an additional 10 ml of 200 proof ethylalcohol, followed by 10 ml of 100% acetone, was added. The sample wasthen centrifuged 1500 rpm for 5 min, the supernatant discarded, and theresidue air dried at room temperature overnight in a hood.

For the glucose oxidase assay, the air-dried sample was analyzed with apurchased kit (Sigma, GAGO-20) that followed the procedure of McClearyet al. (2002). For this assay, 2 ml 2 M potassium hydroxide (KOH) wasadded to the entire dried sample. After 20 min, 8 ml 1.2 M sodiumacetate (pH 3.8) was added, followed by 0.1 ml amyloglucosidase (6,000units/ml; Sigma, A2986). The sample was vortexed and incubated at 50° C.for 30 min. Then the sample was diluted to a total volume of 100 ml withwater and centrifuged at 3000 rpm for 10 min. Resistant starch contentwas determined by adding 3 ml of glucose oxidase solution witho-dianisidine to a 0.1 ml aliquot of the diluted, centrifuged sample.The mixture was incubated at 50° C. for 20 min, and an absorbancereading at 510 nm was recorded. A blank solution (control reading) wasprepared by adding 0.1 ml sodium acetate buffer (0.1 M) to 3 ml glucoseoxidase solution with o-dianisidine, and incubating under the sameconditions as the rice samples. CrystaLean®, untreated rice starch, anduntreated rice flour were also analyzed to use for comparisons. Allanalyses were conducted in duplicate.

Calculation of Resistant Starch

Calculations of resistant starch followed McCleary et al. (2002:RS Yield (%)(samples containing >10% RS):=ΔE×F×(100/0.1)×(1/1000)×(100/W)×(162/180)=ΔE×(F/W)×90RS Yield (%)(samples containing <10% RS):=ΔE×F×(10.3/0.1)×(1/1000)×(100/W)×(162/180)ΔE×(F/W)×9.27

-   -   where, ΔE=absorbance of sample at 510 nm read against a reagent        blank F (conversion from absorbance to micrograms)=100 (μg        glucose)/absorbance of 100 μg glucose    -   W (dry weight of freeze-dried (enzyme-treated) sample)=“as is”        weight×(100−moisture content)    -   100/W=starch as a percent of sample weight    -   162/180=conversion factor, converts free glucose, as determined,        to anhydroglucose as occurs in starch    -   10.3/0.1=volume correction (0.1 mL taken from 10.3 mL) for        samples containing 0-10% RS where the incubation solution was        not diluted, and the final volume is 10.3 mL (McCleary et al.,        2002).        True Yield based on dry weight of 100 g untreated flour or        starch=[RS %×freeze-dried weight of enzyme-treated sample]/(dry        weight of untreated flour or starch)

Example of resistant starch calculation using the sample fromNGNS2hr-pullananse-treated rice starch:F=100/[(1.348+1.382+1.322)/3]=74.07 (based on 3 replicates of the 100 μg/ml glucose standardsolutions)W=100%−6.97%=93.03%RS Yield=0.533×[74.07/(93.03)]×90=38.19%Dry weight of 100 g of untreated rice starch=86.94 gTrue Yield (based on dry weight of untreated rice starch)=(38.19%×67.91g)/86.94 g=29.83%

Statistical Analysis

SAS (Statistical Analysis System) software (version 8.0) was used.Post-hoc multiple comparisons were performed using Tukey's studentizedrange test to test the interactions of incubation period andgelatinization type in enzyme-treated rice flour and starch. The effectsof the treatments on RS yield (RSY), true yield (TY), and freeze-driedsample weight (FDSWt) were examined. The level of significance used wasp≦0.05. Abbreviations used in the following tables and graphs for thevarious samples are: GS for gelatinization with storage, NGNS for nogelatinization and no storage, and GNS for gelatinization and nostorage; rice flour (RF) or starch (RS).

Proximate Analysis

Rice starch and rice flour were analyzed for fat, carbohydrate, protein,and ash content as described in H. J. An, “Properties of OhmicallyHeated Rice Starch and Rice Flour,” Doctorate Thesis, Department of FoodScience, Louisiana State University, Baton Rouge, La. (2001). Samplemoisture was measured as described above, and the results are given inTable 1. All samples were measured in duplicate. As seen in Table 1, themajor differences between rice starch and rice flour were the increasedamount of fat and protein in rice flour. TABLE 1 Proximate analysis ofRice Starch and Rice Flour (% wet basis) Composition (%) Carbo- Samplehydrate Fat Protein Moisture Ash Amylose Commercial 86.06 0.01 0.5613.06 0.31 23.6 Rice Starch White Rice 78.79 0.71 7.77 12.05 0.59 19.4Flour

Example 2

Effect of Pullulanase Treatment on Rice Flour

The results of the treatment and analysis discussed in Example 1 for useof the enzyme pullulanase on rice flour are shown in Table 2. The RScontent of the commercial control, CrystaLean®, was 57.8% (RS Yield,RSY), and 65.7% based on dry weight of untreated CrystaLean® (TrueYield, TY). RSY for the untreated rice flour was 1.32%, and TY was1.50%. The RSY and TY for non-gelatinized rice four treated withpullulanase (NGNS2hr (4.57% RSY, 4.21% TY), NGNS4hr (4.48% RSY, 4.10%TY), and NGNS16hr (3.57% RSY, 3.25% TY)) were not significantlydifferent (Table 2). However, both RSY and TY were significantlydifferent from the commercial control and untreated rice flour (p<0.05).Two of the gelatinized, no storage rice samples, GNS2hr and GNS4hr, had0.95% and 1.24% TY, respectively, and were not significantly differentfrom the untreated rice flour. The other gelatinized samples, (GNS16hr,9.71% TY; GS2hr, 12.7% TY; GS4hr, 10.6% TY, GS16hr, 16.8% TY) weresignificantly different from the commercial control and untreated riceflour (p<0.05). These TY values were approximately one-sixth of thecommercial control, and 6 to 8 times that of the untreated rice flour(Table 2). GS16hr produced both the highest RSY and TY values, 17.3% and16.8%, respectively. TABLE 2 Effects of Gelatinization/Storage andIncubation Duration on Pullulanase Treatments on Rice Flour RS FormationTrue RSYield¹ Yield² Moisture FDSWt³ Sample Treatment (%) (%) (%) (g)Commercial — 57.8a⁴ 65.7a 10 100a   Control Rice Flour — 1.32f 1.50d12.05 100a   Pullulanase NGNS 2 hr 4.57e 4.21d 7.00  81.0bc NGNS 4 hr4.48e 4.10d 5.97  80.5bc NGNS 16 hr 3.57e 3.25d 5.79  80.0bc GNS 2 hr8.67d 0.95d 6.79 11.5e GNS 4 hr 8.67d 1.24d 9.43 12.2e GNS 16 hr 13.6c9.71c 7.50 63.0d GS 2 hr 13.0c 12.75c 9.47 84.0b GS 4 hr 12.8c 10.6c5.99 73.2c GS 16 hr 17.3b 16.8b 11.2 85.2b¹RSYield = Resistant starch yield in percent;²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour);³FDSWt = Sample weight after enzyme treatment followed by freeze-drying;⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

Overall comparison indicated that longer incubation with pullulanaseproduced higher TY values, e.g., TY at GS16hr and GNS16hr as compared tothe 2 hr and 4 hr values. GNS16hr had a similar TY to GS2hr and GS4hr,indicating that overnight storage prior to enzyme treatments couldsubstantially increase RS formation from rice flour.

Non-gelatinized (NGNS) treatments produced TY (3-4%) that were fourtimes higher than that of GNS2hr and GNS4hr (1%) (Table 2). Thegelatinized (GS) samples were 3 to 5 times higher in TY than the NGNSsamples.

Example 3

Effects of α-Amylase Treatment on Rice Flour

For rice flour treated with α-amylase as described in Example 1, theresults are shown in Table 3. The lowest TY was 0.45% from NGNS4hr andGNS16hr (Table 3). GS2hr produced the highest TY at 2.28%. All samplesexcept GS2hr were similar to the untreated rice flour and each other inTY. The TY of all samples was significantly (p≦0.05) lower than thecommercial control. TABLE 3 Effects of Gelatinization/Storage andIncubation Duration on α-Amylase Treatment on Rice Flour RS FormationTrue RSYield¹ Yield² Moisture FDSWt³ Sample Treatment (%) (%) (%) (g)Commercial — 57.8a⁴ 65.7a 10 100a    Control Rice Flour — 1.32f 1.50bc12.05 100a    α-Amylase NGNS 2 hr 4.41e 1.26bc 9.48 22.5bc NGNS 4 hr4.16e 0.45c 8.03  9.40d NGNS 16 hr 3.46e 0.98c 8.98 23.0b  GNS 2 hr6.90cd 0.82c 8.47 10.5cd GNS 4 hr 7.03cd 0.75c 8.54  9.35d GNS 16 hr5.23de 0.45c 8.56 7.6d GS 2 hr 11.6b 2.28b 5.49  17.3bcd GS 4 hr 10.56b1.17bc 7.48  9.80d GS 16 hr 7.23c 1.06c 7.52  13.0bcd¹RSYield = Resistant starch yield in percent calculated (McCleary, 2002)²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour)³FDSWt = Sample weight after enzyme treatment followed by freeze-drying⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

GS2hr had the highest RSY at 11.64% (Table 3), but was not significantlydifferent than GS4hr. However, this RSY was one-fifth of the commercialcontrol. GS16hr was similar to GNS2hr and GNS4hr in RSY, and was 2%higher than GNS16hr. Within the GS treatments, longer incubation timedecreased RSY as GS16hr had 7.23% RSY, while GS2hr and GS4hr hadapproximately 10%. The non-gelatinized (NGNS) treatments had similar RSYand TY values over the time periods. However, NGNS4hr had only 9.4 gFDSWt, which was half of NGNS2hr and NGNS16hr, 22.5 g and 23 g,respectively. The incubation temperature for α-amylase treatments was75° C., which is above the gelatinization temperature of rice flour.This temperature may have facilitated hydrolysis of starch moleculesinto glucose, maltose and α-dextrins, resulting in the low yields.

Example 4

Effects of α-Amylase-Pullulanase Treatment on Rice Flour

For the treatment of rice flour with the enzyme combination ofα-amylase-pullulanase as described in Example 1, the results are shownin Table 4. GS16hr produced the lowest TY, 0.3%, while NGNS2hr andNGNS4hr produced the highest TY at 2.6% (Table 4). NGNS16hr, GNS (2, 4,16 hr), and GS (4, 16 hr) were not different in TY, <1%. TY values forGS2hr and GS4hr were similar to the untreated rice flour at 1.5%. TABLE4 Effects of Gelatinization/Storage and Incubation Duration onα-Amylase-Pullulanase Treatment on Rice Flour RS Formation True RSYield¹Yield² Moisture FDSWt³ Sample Treatment (%) (%) (%) (g) Commercial —57.77a⁴ 65.69a 10 100a   Control Rice Flour — 1.32e 1.50c 12.1 100a  α-Amylase- NGNS 2 hr 3.69d 2.64b 5.96 63b   Pullulanase NGNS 4 hr 4.32d2.62b 6.50 53.5c NGNS 16 hr 3.16d 0.32e 7.50 9f  GNS 2 hr 5.91c 0.61de6.53   9.225f GNS 4 hr 5.97c 0.57e 7.52  8.5f GNS 16 hr 6.10c 0.58e 7.02  8.465f GS 2 hr 7.80b 1.49cd 6.24 16.8d GS 4 hr 7.44b 0.96cde 7.30 11.45e GS 16 hr 5.84c 0.30e 7.19  4.65g¹RSYield = Resistant starch yield in percent calculated (McCleary, 2002)²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour)³FDSWt = Sample weight after enzyme treatment followed by freeze-drying⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

RSYield % increased significantly (p≦0.05) with all enzyme treatmentsover untreated rice flour. However, the TY value only increased slightlyfor two treatments (NGNS2hr and NGNS4hr). Gelatinization did not cause asignificant increase (p>0.05) in TY; however, the FDSWt wassignificantly higher (p≦0.05) in the NGNS2hr and 4 hr samples.

Example 5

Effect of Pullulanase Treatment on Rice Starch

The results of pullulanase treatment of rice starch (as described inExample 1) are given in Table 5. RSY and TY of untreated rice starchwere about 10% of the commercial control (Table 5). The lowest RSY fortreated samples was 12.6% for GNS2hr, and the highest was NGNS4hr at71.5%. These percentages translated to TY values of 3.32% and 61.1%,respectively. For non-gelatinized samples, NGNS4hr was not significantlydifferent from the commercial control. NGNS2hr and NGNS16hr were notsignificantly different from the commercial control, but weresignificantly lower than NGNS4hr in RSY (p≦0.05). For gelatinizedsamples, GNS2hr was significantly lower than GNS16hr in RSY (p≦0.05).GNS16hr had slightly more than double the RSY of GNS2hr, and the TY forGNS16hr was approximately 6 times more than GNS2hr. TABLE 5 Effects ofGelatinization/Storage and Incubation Duration on Pullulanase Treatmentson Rice Starch RS Formation True RSYield¹ Yield² Moisture FDSWt³ SampleTreatment (%) (%) (%) (g) Commercial — 57.8ab⁴ 57.8ab 10.0 100a  Control Rice Starch — 5.39e 5.39ef 13.06 100a   Pullulanase NGNS 2 hr48.7b 41.0c 6.48 77.0b NGNS 4 hr 71.5a 61.1a 7.01 80.0b NGNS 16 hr 48.9b43.3bc 6.51 82.5b GNS 2 hr 12.6de 3.32f 5.98 25.0d GNS 4 hr 14.7cde3.85f 5.61 24.2d GNS 16 hr 29.6c 18.1de 4.73 56.2c GS 2 hr 19.3cde16.1edf 7.03 78.3b GS 4 hr 20.7cde 15.6edf 4.67  69.1bc GS 16 hr 26.8cd22.4d 7.71 78.8b¹RSYield = Resistant starch yield in percent calculated (McCleary, 2002)²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour)³FDSWt = Sample weight after enzyme treatment followed by freeze-drying⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

The gelatinized (GS) treatments were not significantly different(p>0.05) from each other. As the incubation time increased, the RSY andTY increased slightly, with a range of 20-26% RSY and 15-22% TY. AllNGNS and 16 hr treatments resulted in significantly greater RS contentthan the untreated rice starch control (p≦0.05), except for GNS16hr. Thenon-gelatinized samples (NGNS) had higher TY values than any othertreatment, values close to the commercial control. The high TY values inthe NG samples may reflect that the starch granules were more intactthan in the gelatinized samples.

The GNS samples had the lowest FDSWt (24.2 to 56.2 g). The GS ricestarch had significantly higher FDSWt (p≦0.05) than the GNS samples.This was likely due to the overnight refrigeration in the GS sampleswhich allowed the gelatinized starch to retrograde and become moreresistant to enzyme digestion.

Example 6

Effects of α-Amylase Treatment on Rice Starch

For α-amylase treatment on rice starch, the results are shown Table 6.NGNS2hr and NGNS4hr were not significantly different from each other inRSYield. GS4hr had the highest RSY at 70.8%, however the TY was only3.4% due to a low FDSWt, 4.54 g. NGNS2hr and NGNS4hr had similar RSY tothe untreated rice starch. The GNS treatments had the lowest TY, 0.22%,lower than the untreated rice starch. NGNS16hr had the highest TY at14.5%.

The effects of gelling the samples prior to enzyme treatment weresignificant (p≦0.05). The NGNS treatment yielded significantly higherFDSWt (25 to 70 g) and TY (3.18 to 14.5%). The GNS samples had about 1 gof sample left after freeze-drying with 0.2% TY, while GS had 3.32 to8.60 g and 2.24 to 4.37% TY. Gelatinization of rice starch prior toenzyme treatment made the starch granules more accessible to enzymedigestion. TABLE 6 Effects of Gelatinization/Storage and IncubationDuration on α-Amylase Treatments on Rice Starch RS Formation TrueRSYield¹ Yield² Moisture FDSWt³ Sample Treatment (%) (%) (%) (g)Commercial — 57.8b⁴ 57.8a 10.0 100a    Control Rice Starch — 5.39e 5.39c13.06 100a    α-Amylase NGNS 2 hr 7.53e 5.61c 7.68 70.0b  NGNS 4 hr5.03e 3.18de 9.04 60.5c  NGNS 16 hr 56.1bc 14.5b 9.89 25.0d  GNS 2 hr24.5d 0.27f 6.61 1.05h GNS 4 hr 25.2d 0.22f 5.1 0.81h GNS 16 hr 25.0d0.25f 5.75 0.94h GS 2 hr 48.5c 4.37cd 8.00 8.60e GS 4 hr 70.8a 3.41de7.75 4.54f GS 16 hr 67.1a 2.24e 12.3 3.32g¹RSYield = Resistant starch yield in percent calculated (McCleary, 2002)²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour)³FDSWt = Sample weight after enzyme treatment followed by freeze-drying⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

The FDSWt values were higher in the GNS and GS samples for rice flourthan rice starch (Tables 3 and 6), but not in the NGNS rice flour. Thiscould reflect the presence of higher amounts of amylose-lipid complexesin rice flour due to its higher fat content (Table 1). The lower FDSWtin NGNS rice flour may be a result of α-amylase digesting theamylose-lipid complexes present. Table 1 indicates that amylose contentin rice starch (23.6%) was significantly higher than in rice flour(19.4%) (p≦0.05). Tables 3 and 6 also indicate that untreated ricestarch had a higher amount of resistant starch than rice flour.

Example 7

Effect of α-Amylase-Pullulanase Treatment on Rice Starch

The results of treating rice starch with the combination of α-amylaseand pullulanase (as described in Example 1) is given in Table 7. Basedon RSY, NGNS16hr had the lowest yield at 12.51%, while GS16hr had thehighest yield at 52.3%. However, both treatments produced very low TY.NGNS16hr, GNS2hr, GNS4hr, GNS16hr and GS16hr had less than 2 g(freeze-dried sample weight) remaining after enzyme treatment. Thereforeeven though GS16hr had 52.28% RSY, it had only 1.16% TY. NGNS16hr hadonly 0.19% TY. After factoring in the FDSWt, NGNS4hr had the highest TYat 22.9% (Table 7). NGNS16hr, GNS (2, 4, 16 hr), and GS (4, 16 hr) weresignificantly lower than the commercial control, untreated rice starch,and other treatments in TY (p≦0.05). With this enzyme combination,longer incubation time resulted in lower TY and FDSWt. TABLE 7 Effectsof Gelatinization/Storage and Incubation Duration onα-Amylase-Pullulanase Treatments on Rice Starch RS Formation TrueRSYield¹ Yield² Moisture FDSWt³ Sample Treatment (%) (%) (%) (g)Commercial — 57.8a⁴ 57.8a 10.0 100a    Control Rice Starch — 5.39h 5.39c13.06 100a    α-Amylase- NGNS 2 hr 37.3bcde 20.5b 8.03 52.0b Pullulanase NGNS 4 hr 49.7abc 22.8b 7.02 43.0c  NGNS 16 hr 12.5gh 0.19d7.52 1.45f GNS 2 hr 21.6fg 0.41d 4.80 1.75f GNS 4 hr 23.2efg 0.35d 3.831.40f GNS 16 hr 36.6cdef 0.60d 4.32 1.48f GS 2 hr 41.9bcd 5.19c 6.5311.9d  GS 4 hr 30.8def 1.87d 6.45 6.25e GS 16 hr 52.3ab 1.16d 6.49 1.81f¹RSYield = Resistant starch yield in percent calculated (McCleary, 2002)²True Yield = [RSYield × (freeze-dried weight of enzyme treated ricestarch)]/(dry weight of untreated rice flour)³FDSWt = Sample weight after enzyme treatment followed by freeze-drying⁴Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 4 measures.

The gelatinized samples, GS and GNS, had significantly lower FDSWt thanthe NGNS samples, except for NGNS16hr (p≦0.05). The NGNS samples wereapproximately 40 to 50 g higher in FDSWt, and 15 to 20% higher in TY.The GS2hr sample had the highest TY (5.19%) and FDSWt (11.9 g) among theGS and GNS samples; however it was not significantly different from theuntreated rice starch in TY.

Pullulanase is a debranching enzyme and produces oligosaccharides fromstarch molecules, while α-amylase is an endo-enzyme that cleaves α(1,4)linkages randomly to produce glucose, maltose, maltotriose and branchedα-limit dextins. Among the three enzyme treatments for both rice flourand starch, pullulanase alone produced the least breakdown in thestarch. This was visually evident after the enzyme treatments. Theliquid present in the pullulanase treatment flasks was clear andodorless, while the α-amylase and α-amylase-pullulanase treatments hadbrown and sweet smelling liquid. The starch molecules present weredigested by α-amylase to produce simple sugars, thus explaining thelower yield than with pullulanase alone. The α-amylase-pullulanasecombination similarly produced lower yields, especially in thegelatinized samples.

Without wishing to be bound by this theory, it is believed thatpullulanase debranched the starch molecules, making the starch moleculeseven more accessible to α-amylase digestion. Gelatinization of the riceflour and starch resulted in disrupted starch granules making starchmolecules more accessible to enzymes. When gelatinization was combinedwith the synergistic effects of α-amylase and pullulanase, most of thestarch was digested, leaving little resistant starch. In the pullulanasetreatments, the debranching enzyme cleaved the amylopectin branches tocreate linear amylose chains. These chains were allowed to realign andcrystallize into resistant starch since α-amylase was not present tofurther degrade the linear chains. Pullulanase was unable to furtherdegrade the linear chains since it only cleaves α(1,6) linkages.Therefore pullulanase yielded the highest amount of resistant starchamong the three enzyme treatments.

These results indicate that the choice of enzymes and pretreatment ofstarch could affect RS formation. Pullulanase at 4 hr produced thehighest amount of RS among the three enzyme combinations, followed byα-amylase-pullulanase, and then α-amylase. Rice starch had higher RSformation than rice flour, especially in the pullulanase-treatedsamples. Non-gelatinized (NGNS) treatments of rice starch had 40-60% ofTY, while NGNS treatments of rice flour had only 4% TY. The highest TYin rice flour was in the GS pullulanase treatments, a range of 10-17%.Within the pullulanase treatments, the longer incubation treatmentsproduced higher TY.

Pasting Characteristics of Resistant Rice Prepared from Rice Flour andStarch Using Different Enzyme Treatments

When starch is heated past gelatinization in excess water so that thestarch granules swell and become totally disrupted, a viscous mass(paste) is formed. This process is called pasting. The rapid viscoamylograph (RVA) measures the pasting characteristics of starch in rapidvisco units (RVU). The pasting temperature (PT) is the temperature atwhich viscosity of a sample begins to increase. A lower PT indicatesfaster swelling. The peak viscosity (PV) measures the extent ofswelling. During cooking, the starch paste becomes usable once thestarch is heated past PV. The time to peak (TP) is the time required tocook the starch to reach PV. The breakdown (BKD) viscosity is the dropin viscosity from the maximum value (PV) to the minimum value (MV). BKDindicates the stability of the starch paste during cooking, and thecooked paste stability is indicated by final viscosity (FV) at 50° C.The total setback (TSB) is the viscosity increase as the paste is cooledto 50° C. TSB is an indicator of extent of retrogradation of starch.These values were measured by RVA and compared for the resistant ricestarch samples formed in Example 1 from rice flour and rice starch.

Example 8

Materials and Methods for Pasting Experiments

Rapid Visco Amylograph Analysis

Freeze-dried samples of resistant rice starch from Example 1 wereanalyzed by a rapid visco amylograph (RVA) (Newport Scientific, FossFood Technology, Eden Prairie, Minn.). Apparent viscosity of samples wasmeasured in units of RVU (rapid viscosity units), and recorded as afunction of both temperature and time. Procedures for sample preparationwere as directed by the RVA manufacturer. The amount of sample and waterto be used in the RVA analysis was calculated using the followingformulas:S=(88*3.0)/100−MW=25+(3.0−S)

Where,

-   -   S=corrected sample mass (g)    -   W=corrected water volume (mL)    -   M=actual moisture content of the sample (%)

The sample mass and calculated water volume were added to a RVAcanister, and the canister lowered into the RVA. From 0 to 10 sec in theRVA, the temperature was 50° C., and spindle speed was 960 rpm. From 10sec to 1 min, the spindle speed decreased to 160 rpm, but temperatureremained at 50° C. The spindle speed remained at 160 rpm for theremainder of the test. From 1 min to 4:48 min, the temperature increasedlinearly from 50 to 95° C. From 4:48 min to 7:18 min, the temperaturewas held at 95° C. From 7:18 min to 11:06 min, the temperature decreasedlinearly from 95 to 50° C. The temperature remained at 50° C. from 11:06min to 12:30 min, when the test ended. Readings were taken every 4 sec.The idle temperature of the RVA was 50±1° C. Each sample was analyzedtwice using RVA. Peak viscosity (PV), minimum viscosity (MV), finalviscosity (FV), pasting temperature (PT), and time to peak viscosity(TP) were recorded. Set back (SBK), total set back (TSB), and breakdown(BKD) were computed by the following formulas: SBK=FV−PV; TSB=FV−MV; andBKD=PV−MV. All measurements were reported in rapid visco units (RVU).

Statistical Analysis and Sample Abbreviations

SAS (Statistical Analysis System) software (version 8.0) was used.Post-hoc multiple comparisons were performed using Tukey's studentizedrange test to study the interaction of incubation time andgelatinization in each enzyme treatment on rice starch and rice flour.The enzyme treatments were α-amylase (T), pullulanase (P),α-amylase-pullulanase (PT). Incubation periods were 2, 4, 16 hours.Abbreviations were GS for gelatinization with storage, NGNS for nogelatinization without storage, GNS for gelatinization without storage;rice flour (RF) and starch (RS). The level of significance was p≦0.05.

Example 9

Effect of Pullulanase on Pasting Characteristics of Rice Flour

For the pullulanase treated samples, all NGNS (2, 4, 16 hr) treatmentswere significantly different (p≦0.05) from the commercial control in PV,MV, BKD, FV, SBK, TSB and TP (Table 8, FIGS. 1 and 2). As in Example 1,the control was a commercial resistant starch, CrystaLean®. In makingCrystaLean®, the corn starch had been preheated, and therefore indicatedno pasting characteristics in RVA. The CrystaLean® control had 5.75 RVU(PV), 4.42 RVU (MV), and 4.92 RVU (FV). The NGNS samples were treatedwith pullulanase at 55° C. The gelatinization temperature of rice flouris between 70-92° C.; therefore during the pullulanase enzyme treatment,the NGNS-treated samples did not undergo gelatinization. The gelatinized(GNS and GS) samples (FIGS. 3 and 4) had been cooked beforeenzyme-incubation. They were significantly lower in viscosity (p≦0.05)than the NGNS-treated samples and the untreated rice flour. The GNS andGS-treated samples did not have any pasting qualities after their enzymetreatments.

NGNS (2, 4, 16 hr) were not significantly different in PV and MV fromthe untreated rice flour. NGNS2hr had similar breakdown value as theuntreated rice flour (Table 8). NGNS16hr had the highest breakdown (BKD)among the NGNS treatments, 50 RVU higher than untreated rice flour(Table 8). The greater the BKD, the less stable the starch is duringcooking. Thus, NGNS16hr had the lowest cooking stability. It was likelythat the 4 hr and 16 hr incubations had debranched more starch moleculesand reduced their stability in heat. However upon cooling, the FV forNGNS2hr and NGNS4hr increased by 100 and 56 RVU, respectively, andexceeded their PV. NGNS16hr had a FV that was almost identical to itsPV, 233 RVU (Table 8). There was no difference (p>0.05) in SBK, TSB, FV,and TP between the NGNS samples and the untreated rice flour. The TSBvalues suggested that the NGNS samples had less potential forretrogradation than untreated rice flour. The TSB for NGNS ranged from40-90 RVU lower than the untreated rice flour.

The GNS and GS samples had no significant difference from the commercialcontrol in all pasting parameters except TP (Table 8, FIGS. 1, 3, and4). No pasting was observed in these samples or in the commercialcontrol. These samples had been gelatinized prior to enzyme treatment bycooking at 95° C. The BKD, SBK and TSB values were low as little to noincrease in viscosity occurred during the RVA test.

The PT for NGNS4hr and NGNS16hr was at 83.6° C., while the PT foruntreated rice flour and NGNS2hr was 86° C. The NGNS samples tookslightly less than 6 min to cook, just like untreated rice flour. TheGNS and GS samples were reported to cook at 3.7 to 6.3 min. However,when referring to the RVA thermograms, neither a pasting peak nor a PTwas found (Table 8).

The incubation time did not significantly affect the pastingcharacteristics of any sample. (Table 8). On the other hand,gelatinization type had an effect on the pasting characteristics. TABLE8 Effects of Gelatinization/Storage and Incubation Duration on PastingCharacteristics of Pullulanase Treated Rice Flour^(1,2,3) SampleTreatment PV MV BKD FV SBK TSB TP PT Control — 5.75b³ 4.42b ND 4.92b NDND 2.2811d ND Rice Flour — 235.83a 154.63a 81.21c 377.21a 141a   222.58a 5.4913abc 86.5a Pullulanase NGNS 2 hr 227.50a 135.06a 92.44c318.63a 91.1ab 183.56a 5.9312abc 86.7a NGNS 4 hr 247.54a 135.77a 111.77b303.6a 56.1ab 167.83a 5.9151abc 83.63b NGNS 16 hr 223.63a 92.04a 131.58a223.02a −0.60b 130.98a 5.7031abc 83.81b GNS 2 hr 6.35b 4.29b 1.88d 4.81bND ND 3.7061cd ND GNS 4 hr 4.9b 3.63b 1.10d 4.4b ND ND 3.9263bcd ND GNS16 hr 6.83b 5.29b 1.63d 8.73b  1.90b 3.44b 6.4278a ND GS 2 hr 7.64b6.75b 0.89d 9.64b  2.00b 2.89b 5.8659abc ND GS 4 hr 8.71b 7.58b 1.13d11.21b 2.5b 3.62b 5.8168abc ND GS 16 hr 6.75b 5.33b 1.5d 7.42b ND 2.08b6.2895ab ND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; ND = non-detectable.²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.

Example 10

Effect of α-Amylase Treatment on Pasting Characteristics of Rice Flour

No significant pasting was seen in any of the treatments. Even thenon-gelatinized (NGNS) samples had little pasting qualities due to thehigh temperature (75° C.) of incubation with α-amylase, a temperaturethat will gelatinize flour (Table 9; graphs of viscosity not shown).There were no significant differences in the PV, MV, BKD, FV, SBK, TSB,and TP among the NGNS, GNS, and GS treatments and the commercial control(Table 9).

The NGNS2hr and 16 hr samples had a pasting peak at 4.4 min with PV of7.56 and 8.61 RVU. The BKD values were 4.61 and 5.06 RVU, respectively,indicating some breakdown in viscosity during the cooking process. ForNGNS4hr, no increase in viscosity was seen during cooking. It is unclearwhy the NGNS2hr and 16 hr treatments, one shorter and one longer thanNGNS4hr, had small pasting peaks. The TP for all the treatments rangedfrom 2.48 to 6.62 min. Only the NGNS2hr and 16 hr enzyme-treated sampleshad peaks large enough to indicate a breakdown. The rest of the sampleshad already been cooked and PT was not detected.

The α-amylase treated rice flour would not be suitable as an ingredientin viscous food products due to the minimal pasting characteristics.TABLE 9 Effects of Gelatinization Storage and Incubation Duration onPasting Characteristics of α-amylase Treated Rice Flour^(1,2,3) SampleTreatment PV MV BKD FV SBK TSB TP PT Control —  5.75b³ 4.42cd ND 4.92cdeND ND 2.28b ND Rice Flour — 236a    155a     81.2a  377a     141a   223a    5.49ab 86.5a α-Amylase NGNS 2 hr 7.56b 2.94d  4.61b 3.56e −4.00b ND 4.42ab ND NGNS 4 hr 5.17b 3.71cd ND 4.00ed  ND ND 4.45ab NDNGNS 16 hr 8.61b 3.56cd 5.06b 4.03ed  −4.58b ND 4.41ab ND GNS 2 hr 5.15b4.25cd ND 4.79cde ND ND 5.17ab ND GNS 4 hr 4.83b 4.25cd ND 4.81cde ND ND5.30ab ND GNS 16 hr 5.53b 4.92cd ND 5.94bcd ND 1.03b 6.62a ND GS 2 hr8.88b 7.17b  ND 8.04b  ND ND 2.48ab ND GS 4 hr 7.29b 5.58bc ND 6.5bc  NDND 6.26ab ND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; ND = non-detectable.²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.

Example 11

Effect of α-Amylase-Pullulanase Treatment on Pasting Characteristics ofRice Flour

For the samples incubated with both α-amylase and pullulanase, nosignificant difference was found between the commercial control and theNGNS samples in pasting characteristics in PV, MV, BKD, FV, SBK, TSB andTP (Table 10, viscosity figures not shown). The temperature for enzymeincubation was 60° C. for all treatments. No significant differences wasseen between the gelatinized samples and the commercial control. NGNS2hrand NGNS4hr samples had 5.6 and 6.8 RVU in BKD, the only detectable BKDbesides the native rice flour. Since the GNS and GS samples were alreadygelatinized, no pasting temperatures were detected. The NGNS2hr andNGNS16hr samples also had no pasting temperatures, and only onereplicate of NGNS4hr had a PT at 81.5° C. Since only one NGNS4hrreplicate showed pasting, the average reported in Table 10 is distorteddue to dividing 81.5° C. by four replicates.

The NGNS2hr and 4 hr samples had significantly lower pasting abilitiesthan untreated rice flour (p≦0.05) (Table 10). The NGNS samples had apeak at 3.75 and 3.88 min with PV at 10.9 and 11.5 RVU, respectively.There was a small amount of breakdown as measured by MV being one-thirdof PV for the two samples.

The α-amylase-pullulanase treated NGNS samples had significantly lowerpasting qualities than the NGNS samples treated with only pullulanase.It is possible that the higher incubation temperature for theα-amylase-pullulanase treatment was responsible for part of thedifference. It is also possible that α-amylase degraded the starch sothe sample could not paste like untreated rice flour. The NGNS16hr, GNSand GS samples were almost identical to each other in the RVA analysis,but were significantly different from the untreated rice flour (Table10). None of these samples had an increase in viscosity, and thereforeno BKD, SBK and TSB was detected. Due to the lack of pasting in thesesamples, they are not suitable for food products that are highly viscous

The post-cooking viscosity of all NGNS samples was not significantlydifferent from the commercial control. There was no increase in FV inany treated samples, whereas the FV for untreated rice flour was thehighest reading among all samples. The only reported PT was for NGNS4hr.The TP for all treatments were not significantly different from thecommercial control and untreated rice flour. The time to peak valueswere between 2.25 to 5.82 min. TABLE 10 Effects ofGelatinization/Storage and Incubation Duration on PastingCharacteristics of α-Amylase-Pullulanase Treated Rice Flour^(1,2,3)Sample Treatment PV MV BKD FV SBK TSB TP PT Control —  5.75bc 4.42b ND4.92b ND ND 2.28a ND Rice Flour — 236a    155a    81.2a 377a    141a   223a 5.49a 86.5a α-Amylase- NGNS 2 hr 10.9bc  3.88b 5.60bc 4.42b −6.50cdND 3.75a ND Pullulanase NGNS 4 hr 11.5b  3.79b 6.83b 4.27b −7.25d  ND3.88a 20.4a NGNS 16 hr 4.88c 4.00b ND 4.46b ND ND 3.60a ND GNS 2 hr5.25c 4.42b ND 4.92b ND ND 4.19a ND GNS 4 hr 5.08c 4.71b ND 5.33b ND ND5.82a ND GNS 16 hr  5.92bc 5.08b ND 5.21b ND ND 3.89a ND GS 2 hr 5.29c3.54b ND 4.29b ND ND 2.25a ND GS 4 hr  5.92bc 4.79b ND 5.21b ND ND 5.44aND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; and ND = non-detectable.²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.

Example 12

Effects of Pullulanase Treatment on Pasting Characteristics of RiceStarch

For pullulanase-treated rice starch, the pasting activity in the NGNS(2, 4, 16 hr) samples was similar (Table 11), but was significantlygreater (p≦0.05) than the commercial control. NGNS treatment increasedthe PV by 30-48 RVU as compared to untreated rice starch, with theNGNS4hr sample having the greatest difference at 48 RVU (FIGS. 1 and 5).There was twice as much BKD in the NGNS samples than in the untreatedrice starch. Pasting temperature was in a similar range, approximately80° C. Within the NGNS treatments, the 16 hr treatment had the lowestSBK and TSB, 30 to 40 RVU lower than the 2 hr and 4 hr treatments.

The GNS2hr and 4 hr treatments showed some pasting activity (Table 11,FIG. 6). There was a decrease in PV, MV and FV as the incubation timeincreased. GNS4hr had the highest BKD (54.54 RVU) and PT (89.62° C.)among the three GNS treatments.

There was a progressive decrease in pasting properties among the NGNS,GNS and GS samples. (FIGS. 5, 6, and 7) The NGNS samples had the highestpasting characteristics, similar to that of the untreated rice starch.The NGNS samples had slightly higher values for PV, MV, and FV ascompared to the untreated rice starch. GNS16hr and the GS treatmentswere significantly lower (p≦0.05) than the untreated rice starch in PV,MV, FV, BKD, SBK and TSB (Table 11; FIGS. 1, 6, and 7). The SBK and TSBof the GNS and GS treated samples were not significantly different fromthe commercial control. GS2hr and GS4hr had 6.33 and 2.63 RVU in BKD,and 6.94 and 3.15 RVU in TSB, respectively. SBK was not detected. TheGNS-treated samples had decreasing pasting characteristics as incubationtime increased, and were significantly lower than the untreated ricestarch (Table 11, FIGS. 1 and 6). The GNS2hr sample had the best pastingproperties among the three GNS samples, followed by GNS4hr and thenGNS16hr. The FV values for all three samples were lower than the PV. TheBKD for the NGNS sample was higher than the untreated rice starch, 23 to31 RVU higher. GNS16hr had no pasting activity and had no breakdown, andwas similar to the GS treatments. The general trend of all thepullulanase treatments was the longer the incubation, the lesser thepasting qualities.

The GS treated rice starch had significantly less pasting than theuntreated rice starch. The highest PV was only 22.8RVU in the GNS2hrsample. BKD ranged from 0 to 6.33 RVU, SBK was not detectable, and TSBwas 3.15 to 6.94 RVU.

The NGNS samples had the highest retrogradation potential as they hadthe largest TSB and SBK values while GNS had the lowest. The NGNS andGNS2hr and 4 hr samples also had the highest BKD values indicating agreater disruption of starch granules during cooking.

The treated rice flour took a shorter time to cook than the treated ricestarch. The highest TP was 6.25 min in the NGNS16hr sample. The NGNS,and GNS2hr samples had similar PT (p>0.05, Table 11) to untreated riceflour, 79 to 84.5° C. The GNS2hr and GNS4hr samples were notsignificantly different from each other in PT, 84.5 and 89.6° C.,respectively. The GNS16hr and GS samples showed no pasting, and were notsignificantly different from the commercial control. TABLE 11 Effects ofGelatinization/Storage and Incubation Duration on PastingCharacteristics of Pullulanase Treated Rice Starch^(1,2,3) SampleTreatment PV MV BKD FV SBK TSB TP PT Control — 5.75d 4.42c  0d 4.92c  0c 0c 2.28f ND Rice Starch — 198.13b 174.67a 23.46bc 271.08b  73.0a 96.4ab 6.32abc 81.23b Pullulanase NGNS2 hr 234.73ab 186.67a 48.06a296.44ab  61.7a 110a 6.07bc 79.03b NGNS4 hr 246.9a 196.13a 50.770a316.15a  69.3a 120a 6.11bc 80.14b NGNS16 hr 232.02ab 189.29a 42.73ab265.52b  33.5b  76.2b 6.25bc 81.55b GNS2 hr 88.83c 42.31b 46.52ab 46.94c−41.9d  3.63c 5.87cd 84.54ab GNS4 hr 82.73c 28.19bc 54.54a 29.98c −52.8d 0c 6.42ab 89.62a GNS16 hr 8.33d 6.79c  0d 7.92c  0c  0c 6.84a ND GS2 hr22.81d 15.67bc  6.33cd 22.60c  0c  6.94c 3.76e ND GS4 hr 12.83d 10.21c 2.63cd 13.35c  0c  3.15c 5.40d ND GS16 hr 8.6d 7.69c  0d 8.27c  0c  0c6.78a ND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; and NO = non-detectable.²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.

Example 13

Effects of α-Amylase Treatment on Pasting Characteristics of Rice Starch

There was insufficient sample in the GS (all) and GNS (4, 16 hr)treatments to conduct RVA analysis. The GS2hr and NGNS (4, 16 hr)treated samples were not significantly different from the commercialcontrol in PV, MV, FV, SBK, TSB and PT (Table 12; no viscosity figuresshown). The NGNS samples had pasting activity at 80 to 84° C., and tookabout 3.9 to 4.4 min to reach the pasting peak. This time is less thanthe TP for untreated rice starch, 6.3 min (p≦0.05). The BKD for theNGNS2hr sample was greater than the untreated rice starch (p≦0.05),while the NGNS4hr and 16 hr samples were not significantly greater thanthe untreated rice starch in BKD (Table 12). There was considerableamount of BKD in the NGNS treated samples as the MV and FV were 4.6 to6.58 RVU. The untreated rice starch had better cooking stability thanthe NGNS treated samples. The NGNS and GNS2hr treated samples had SBKsfrom −95.3 to 73 RVU, and no TSB since the FV was much lower than the PV(Table 12). The GNS2hr sample did not have a PT, probably because thesample had been gelatinized prior to enzyme treatment. TABLE 12 Effectsof Gelatinization/Storage and Incubation Duration on PastingCharacteristics of α-Amylase Treated Rice Starch^(1,2,3) SampleTreatment PV MV BKD FV SBK TSB TP PT Control — 5.75c 4.4167b  0b 4.917b 0b 0b 2.2811d ND Rice Starch — 198.13a 174.6667a 23.46b 271.083a  73.0a 96.42a 6.3224a 81.225a α-Amylase NGNS2 hr 100.98b 4.1875b 96.79a 5.67b−95.3c 0b 4.1225b 80.663a NGNS4 hr 67.13bc 3.8550b 63.27ab 4.605b−62.5bc 0b 3.8850b 80.35a  NGNS16 hr 69.46bc 4.415b 65.04ab 6.583b 62.9bc 0b 4.4325b 84.113a GNS2 hr 7.67c 3.5b  3.46b 4.667b  −3.00b 0b3.1278c ND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; and ND = non-detectable²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.

Example 14

Effects of α-Amylase-Pullulanase Treatment on Pasting Characteristics ofRice Starch

There was insufficient sample from NGNS16hr, GNS (all) and GS16hrtreatments to collect pasting characteristic data on them. All thevariables except BKD examined for GS and NGNS treatments weresignificantly lower than values for the untreated rice starch, but weresimilar to the commercial control (Table 13, viscosity figures notshown). There was no breakdown, setback, and total setback in the GS2hrand GS4hr samples. The NGNS2hr and NGNS4hr samples had 18 RVU for PV, 3RVU for MV and 14 RVU for BKD (Table 13). The NGNS samples showed lessbreakdown than in the untreated rice starch. All the samples had verylow FV, which resulted in 0 values for TSB.

The effects of α-amylase-pullulanase treatment on rice flour and starchwere very similar. Although the samples had very low or zero values forSBK, TSB and BKD which indicates stability during cooking (Tables 10 and13), these products would not be recommended for use in viscous foodproducts because the pasting viscosities were low. TABLE 13 Effects ofGelatinization/Storage and Incubation Duration on PastingCharacteristics of α-Amylase-Pullulanase Treated Rice Starch^(1,2,3)Sample Treatment PV MV BKD FV SBK TSB TP PT Control — 5.75b 4.42b  0b4.92b  0b 0b 2.28d ND Rice Starch — 198a    175a    23.5a 271a     73.0a 96.4a 6.32a 81.2a α-Amylase-Pullulanase NGNS2 hr 18.2b  3.52b 14.6ab4.13b −14.0b 0b 3.83b 60.6a NGNS4 hr 17.4b  3.71b 13.7ab 4.40b −13.0b 0b3.99b 40.7a GS2 hr 5.83b 3.08b  0b 3.58b  0b 0b 2.44c ND GS4 hr 6.25b5.13b  0b 5.63b  0b 0b 6.45a ND¹Abbreviations: PV = Peak Viscosity; MV = Minimum Viscosity; BKD = Breakdown; FV = Final Viscosity; SBK = Set back; TSB = Total Set Back; TP =Time to Peak; PT = Pasting Temperature; and ND = non-detectable.²Units: Viscosity (RVU); Temperature (° C.); Time (min)³Means with different letters in each column are significantly different(p ≦ 0.05). The values are an average of 4 measurements.

In rice starch, the effects of the enzymes treatments on the pastingproperties were complex due to the different temperatures used duringincubation. The target linkages of pullulanase and α-amylase alsoresulted in different end products. Pullulanase debranches the starchmolecules, while α-amylase cleaves randomly within the starch molecules.The longer chained molecules left from pullulanase debranching were ableto paste better as observed in the RVA analysis. The α-amylase-treatedsamples had little pasting properties due to random cleaving of thestarch molecules, in addition to the higher incubation temperature (75°C.) required for optimum enzyme activity. The high incubationtemperature was within the range of starch gelatinization temperatures.Therefore during RVA analysis, the α-amylase samples did not displaysignificant pasting properties even when the sample had not beengelatinized prior to enzyme treatment. The α-amylase-pullulanase sampleshad very similar pasting properties to the α-amylase treated samples,probably due to the synergistic effects of α-amylase and pullulanase indigesting the starch molecules. Most of the starch molecules had beendegraded into simple sugars which do not paste like untreated rice flourand starch.

Due to the absence or low availability of lipids and proteins, theuntreated rice starch had slightly different pasting properties thanuntreated rice flour. There was a greater potential for retrogradationin rice flour as seen in FIG. 5, probably due to the presence of lipids.However, in GNS-pullulanase treated rice flour, there was virtually noincrease in viscosity during cooking or holding temperature, while thesame treatment on rice starch produced significant pasting upon cookingand retrogradation during storage (FIGS. 3 and 6). In the GS-pullulanasetreated rice flour, the PV, MV, FV and TSB were not as pronounced as theGS-pullulanase treated rice starch.

In the NGNS treated rice starch and flour, the pullulanase-treatedsample had the best pasting properties. The α-amylase treatment on NGNSrice starch resulted in greater PV values than the α-amylase-pullulanasetreated NGNS rice starch. However, both had no cooking stability as theMV values were similar to the viscosity detected prior to PV. The NGNSrice flour and rice starch treated with α-amylase andα-amylase-pullulanase were very similar in pasting properties. There waspasting observed during heating, however the peak rapidly disappeared asthe temperature was held at 95° C.

The pasting qualities of both rice starch and flour samples changedaccording to enzyme and incubation temperature. Samples treated withpullulanase, regardless of gelatinization and storage state, had higherPV, MV and FV, probably because these samples were incubated at 55° C.,thus not exceeding the pasting temperature of 60-78° C. In addition,pullulanase is a debranching enzyme as opposed to α-amylase, whichrandomly cleaves α(1,4) glycosidic bonds. There was greater degradationin the samples when α-amylase was used; both α-amylase andα-amylase-pullulanase treatments had lower pasting qualities. Thesamples that were gelatinized displayed little or no pasting qualitiesas expected. The NGNS rice starch samples treated with pullulanase hadslightly higher PV, FV and BKD than the untreated rice starch. The NGNSpullulanase treated rice flour samples had very similar pastingqualities as the untreated rice flour.

Only the NGNS samples on flour and starch that were treated withpullulanase retained their pasting characteristics. Resistant starchproduced by this method would be more suitable for manufacturing foodproducts with high viscosity after cooking than the commercialCrystaLean®.

Heating Profiles of Enzyme Treated Rice Starch and Rice Flour AsDetected by Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) measures heat absorbed or givenoff by a sample in a controlled atmosphere at specified temperatures.DSC provides information about a specific heat and latent heat ofsamples as temperature rises, which indicates changes in the amorphousand crystalline structures. Data is recorded in terms of heat flow, andis presented in joules/gram (J/g). (Cassel, 2002) In the analysis ofstarch, starch gelatinization parameters such as peak onset, peaktemperature, end of peak, and gelatinization enthalpy information iscollected. DSC can detect the presence of resistant starch in samples.RS was found to give endothermic peaks between 136 to 162° C., whileamylose-lipid complexes exhibited peaks at 95-130° C. (Sievert andPomeranz, 1989).

Example 15

Differential Scanning Calorimetry (DSC) Materials and Methods

Samples of resistant starch were prepared as described in Example 1.Differential Scanning Calorimetry (DSC) was conducted to measurespecific and latent heat which indicates structural changes fromamorphous to crystalline. DSC was measured in a model Q100, TAInstruments (New Castle, Del.). DSC Pans were purchased from TAInstruments (Part no. 900825.902, T21230; New Castle, Del.). A 10 mgsample was placed in the pan, and 20 mg water added. The pan was sealed,and the samples equilibrated overnight at room temperature. Duringanalysis, the sample was heated at 35° C. for five minutes, and thenheated to 140° C. at a rate of 5° C./min. Samples that indicated peaksbeyond the gelatinization temperature range, 60-80° C., were reheated todetermine the stability of the peaks. Four replicates were analyzed foreach treatment.

Statistical analyses were conducted using SAS software (version 8.0) asdescribed in Example 1. Post-hoc multiple comparisons were performedusing Tukey's studentized range test to test the interactions ofincubation periods and gelatinization type in enzyme treated rice flourand starch, and the effects on peak onset, peak, and end of peaktemperatures. Abbreviations for sample preparation are as describedabove: GS for gelatinization with storage, NGNS for no gelatinizationwithout storage, GNS for gelatinization without storage, RF for riceflour, and RS for rice starch.

Example 16

Effects of Pullulanase Treatment on Heating Profile of Rice Flour

The commercial control, CrystaLean®, was analyzed by DSC along with thesamples and untreated rice flour. The commercial control had nogelatinization activity at normal temperatures, indicating priorgelatinization (Table 14, FIG. 8).

The NGNS-treated samples were not significantly different from theuntreated rice flour for gelatinization, amylose-lipid complex, andresistant starch peaks (Table 14; FIGS. 8 and 9). For the untreated riceflour, the enthalpy for the gelatinization peak was 2.85 J/g. Theenthalpy required to produce the gelatinization peak in the NGNS treatedsamples was 10 to 15 times higher than the untreated rice flour (Table14). A higher peak enthalpy means that a greater amount of energy wasrequired to produce the peak. The starch granules within the sample maybe more compact and more resistant to cooking. There was no significantdifference in the other treated samples and the untreated rice flour inpeak onset, peak, and completion temperatures, and enthalpy. Thetemperature ranges for gelatinization peak onset was 49.0 to 71.9° C.,peak was 57 to 81.1° C. and completion was 69.6 to 98.0° C. (Table 14).The enthalpy range was between 0.18 and 41.9 J/g.

For the amylose-lipid complex peak, the peak onset temperatures rangedfrom 76.3 to 101° C. The commercial control did not have a peak. Thepeak onset temperature for the GNS16hr treated sample was significantlylower (p≦0.05) than the untreated rice flour (FIGS. 8 and 10; Table 14).The GNS2hr and 4 hr treated samples had significantly higher (p≦0.05)peak onset temperatures than GNS16hr and GS16hr. No significantdifference was seen in the completion temperatures and peak enthalpiesbetween any sample and the untreated rice flour. TABLE 14 Effects ofGelatinization/Storage and Incubation Duration on ThermalCharacteristics of Pullulanase Treated Rice Flour^(1,2,3) FirstTransition Second Transition Third Transition (Gelatinization)(Amylose-lipid complex) (Resistant Starch) Sample Treatment T_(o) T_(p)T_(C) ΔH T_(o) T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH Control — ND ND ND NDND ND ND ND 101a 119a 133a 3.69a Rice Flour — 70.9a 77.8a 92a   2.85a 94.2ab 100abc 106a 0.184a 118a 119a 128a 0.172b Pullulanase NGNS2 hr68.9a 76.2a 90.7a 31.64a  98.0a 103ab 110a 2.19a 124a 126a 129a 0.519bNGNS4 hr 63.0a 69.9a 90.3a 38.7a  91.9abc  98.4abc 107a 1.45a ND ND NDND NGNS16 hr 63.4a 69.7a 88.6a 41.9a  93.8abc 101abc 111a 12.8a 120a121a 127a 0.0159b GNS2 hr 51.8a 61.5a 70.5a 0.51a 101a 109a 121a 1.03aND ND ND ND GNS4 hr 71.9a 81.1a 91.8a 0.44a 101a 110a 123a 0.896a ND NDND ND GNS16 hr 51.3a 74.3a 98.0a 4.15a  78.3bc  90.1bc 107a 1.50a 114a120a 130a 0.235b GS2 hr 49.0a 60.9a 83.9a 3.43a  89.3abc  97.3abc 108a0.801a 111a 114a 122a 0.187b GS4 hr 54.9a 62.2a 77.6a 0.75a  88.4abc 95.1bc 107a 0.652a 108a 113a 124a 0.363b GS16 hr 51..2a 57.0a 69.6a0.18a  76.3c  88.1c 105a 2.78a 106a 111a 119a 0.131b¹T_(o,) T_(p,) T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

For the resistant starch peak, no significant difference were found inthe commercial control, untreated rice flour and all the treated samplesfor peak onset, peak and completion temperature. For the NGNS4hr,GNS2hr, and GNS4hr treated samples, no resistant starch peak wasdetected (FIGS. 9, 10, and 11). The resistant starch peak in thecommercial control, CrystaLean® (100 to 130° C.), appeared to be twopeaks overlapping each other so that the start and end points of thepeaks were not distinct (FIG. 8). According to the DSC results, theNGNS2hr and 16 hr, GNS16hr, and GS treated samples contained resistantstarch (FIGS. 8, 9, 10, and 11; Table 14).

Example 17

Effects of α-Amylase Treatment on the Heating Profile of Rice Flour

On the α-amylase-treated samples, the NGNS2hr and 4 hr, GNS2hr, andGS4hr samples did not have a gelatinization peak. The onset, peak andcompletion temperatures of the NGNS16hr, GNS4hr and 16 hr, and GS2hr and16 hr treated samples were not significantly different from theuntreated rice flour in gelatinization temperatures and peak enthalpies(Table 15, heating profile graphs not shown).

The onset temperature range for the amylose-lipid complex peak was 82.2to 104° C. (Table 15). The peak onset temperature for NGNS4hr wasapproximately 20° C. lower than the GNS16hr, GS4hr and GS16hr treatedsamples, a significant difference (p≦0.05). No significant differenceswere found in enthalpies for all the treated samples and the untreatedrice flour (Table 15).

The resistant starch peak for the commercial control had an enthalpy of3.69 J/g. The highest peak enthalpies were observed in the GS2hr andGS4hr treated samples, 125 and 108 J/g, respectively (Table 15). Theywere significantly higher in peak onset temperature than the commercialcontrol (p≦0.05). Incubation time within the NGNS treatments did notproduce a significant difference in peak enthalpy (Table 15). Nosignificant differences were detected in the treated samples, commercialcontrol, and untreated rice flour in the resistant starch peaktemperature.

Based on the DSC analysis, 3 α-amylase-treated samples (GNS2hr and 16hr, and GS16hr) did not contain resistant starch. However,gelatinization type and incubation period did not effect peak onset,peak, and completion temperatures. TABLE 15 Effects ofGelatinization/Storage and Incubation Duration on ThermalCharacteristics of α-Amylase Treated Rice Flour^(1,2,3) First TransitionSecond Transition Third Transition (Gelatinization) (Amylose-lipidcomplex) (Resistant Starch) Sample Treatment T_(o) T_(p) T_(C) ΔH T_(o)T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH Control — ND ND ND ND ND ND ND ND101b 119a 133a 3.69a Rice — 70.9a 77.8a 92.0a 2.85a   94.2ab 100ab 106a0.18a 118ab 119a 128a 0.17a Flour α- NGNS2 hr ND ND ND ND  88.3ab 94.5ab 108a 4.06a 108ab 112a 118a 0.86a Amylase NGNS4 hr ND ND ND ND 82.2b  85.2b  94.5a 0.26a 102b 111a 129a  0.191a NGNS16 hr 71.0a 75.0a86.0a 1.48a   96.3ab 104ab 114a 1.06a 108ab 114a 120a 0.14a GNS2 hr NDND ND ND  99.8ab 109ab 126a 1.82a ND ND ND ND GNS4 hr 58.6a 66.0a 78.1a0.121a  90.0ab  97.0ab  88.4a 26.4a 106ab 119a 128a 2.02a GNS16 hr 59.7a83.1a 84.9a 0.664a 104a 110a 126a 0.89a ND ND ND ND GS2 hr 67.8a 71.0a97.4a 1.84a   98.9ab 105ab 114a 0.46a 122a 126a 134a 125a    GS4 hr NDND ND ND 101a 110a 120a 2.52a 120a 126a 134a 108a    GS16 hr 64.6a 67.4a77.1a 0.226a 100a 108ab 121a 1.55a ND ND ND ND¹T_(o), T_(p), T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measurements.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

Example 18 Effects of α-Amylase-Pullulanase Treatment on the HeatingProfile of Rice Flour

For samples treated with both α-amylase and pullulanase, agelatinization peak for the NGNS16hr, GNS4hr and 16 hr, and GS2hr and 16hr samples was not detected. No significant differences were seen in thegelatinization peak onset, peak, and completion temperatures between theNGNS2hr and 4 hr, GNS2hr and GS4hr treated samples and the untreatedrice flour (Table 16, no heating profile graphs shown). The peakenthalpies for the GNS2hr (0.144 J/g) and GS4hr (0.602 J/g) treatedsamples were significantly lower than that of the untreated rice flour(2.85 J/g) (p≦0.05).

For the GS2hr treated sample, the amylose-lipid complex peak onsettemperature was significantly lower (p≦0.05) than that of the untreatedrice flour. The peak enthalpy for the GS2hr (1.84 J/g) treated samplewas significantly higher than the NGNS2hr and 4 hr treated samples (0.15J/g), and untreated rice flour (0.18 J/g) (p≦0.05).

Neither the NGNS16hr nor all GNS treatments resulted in a resistantstarch peak. NGNS2hr and 4 hr and all the GS treatments were similar toboth the commercial control and untreated rice flour in peak onset, peakand completion temperatures, and peak enthalpies.

Incubation time did not have a significant effect on the gelatinizationtype (Table 16). The enzyme treatments did not produce a significantamount of resistant starch as shown in Table 4; however, the smallamount present in the samples was detected by DSC. The non-significantdifferences between the wide ranges of temperature within each variable,peak onset, peak, and completion temperatures were due to inconsistentresults during analysis. TABLE 16 Effects of Gelatinization/Storage andIncubation Duration on Thermal Characteristics of α-Amylase-PullulanaseTreated Rice Flour^(1,2,3) First Transition Second Transition ThirdTransition (Gelatinization) (Amylose-lipid complex) (Resistant Starch)Sample Treatment T_(o) T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH T_(o) T_(p)T_(C) ΔH Control — ND ND ND ND ND ND ND ND 101a 119a 133a 3.69a RiceFlour — 70.9a 77.8a 92.0a 2.85a 94.2a 100ab 106a 0.184b 118a 119a 128a0.17a α- NGNS2 hr 71.7a 78.2a 93.6a 3.04a 95.5a 101ab 108a 0.157b 115a119a 126a 0.09a Amylase- NGNS4 hr 73.3a 77.8a 91.6a 3.18a 92.2a  95.7ab102a 0.157b 105a 111a 121a 0.56a Pullulanase NGNS16 hr ND ND ND ND 97.1a106a 118a 1.23ab ND ND ND ND GNS2 hr 57.7a 64.2a 77.8a  0.144b 97.7a106a 119a 1.24ab ND ND ND ND GNS4 hr ND ND ND ND 100a   109a 123a 1.43abND ND ND ND GNS16 hr ND ND ND ND 97.6a 105a 119a 1.05ab ND ND ND ND GS2hr ND ND ND ND 73.9b  86.8b 101a 1.84a 109a 115a 124a 117a    GS4 hr58.7a 67.9a 79.7a  0.602b 99.5a 104a 111a 1.21ab 106a 117a 123a 1.52aGS16 hr ND ND ND ND 94.9a 102ab 113a 1.41ab 113a 122a 133a 53.8a ¹T_(o,) T_(p,) T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

Example 19

Effects of Pullulanase Treatment on Heating Profile of Rice Starch

For the samples incubated with pullulanase, the NGNS-treated samples hadgelatinization characteristics similar to the untreated rice starch(Table 17; FIGS. 8 and 12). The peak enthalpy was also not differentfrom the untreated rice starch. The NGNS treated samples were notsignificantly different from each other. The GNS-treated samples did notshow a gelatinization peak (Table 17; heating profile graphs not shown),which was expected since the samples had been gelatinized at 95° C.prior to enzyme treatment. However, the GS-treated samples had anunexpected gelatinization peak and were not significantly different fromthe untreated rice starch (Table 17). The enthalpies of the GS2hr and 16hr treated samples, however, were significantly lower (p≦0.05) than thatof the untreated rice starch and the NGNS treated samples. The peakonset range was 62.8 to 73.1° C., peak range was 74.2 to 81.3° C., andcompletion range was 85.5 to 96.7° C.

No significant difference was found in amylose-lipid complex peak onset,peak, and completion temperatures, and enthalpies in the treated samplesand the untreated rice starch (Table 17). The peak onset range was 69.2to 99.7° C., peak range was 73.6 to 121° C., and completion range was79.4 to 121° C.

A resistant starch peak was not detected in the NGNS16hr treated sample.No significant differences were found in peak onset and peaktemperatures between the other treated rice starch samples, untreatedrice starch, and commercial control (p>0.05). The peak completiontemperature in GS16hr was significantly higher (12° C.) than theuntreated rice starch (p≦0.05). The enthalpies ranged from 0.00835 to0.3 J/g (Table 17). The peak onset range was 96.8 to 120° C., peak rangewas 120 to 125° C., and completion range was 125 to 133° C. TABLE 17Effects of Gelatinization/Storage and Incubation Duration on ThermalCharacteristics of Pullulanase Treated Rice Starch^(1,2,3) FirstTransition Second Transition Third Transition (Gelatinization)(Amylose-lipid complex) (Resistant Starch) Sample Treatment T_(o) T_(p)T_(C) ΔH T_(o) T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH Control — ND ND ND NDND ND ND ND 101a 119a 132a 3.6b Rice — 68.8ab 73.3a 83.6b 2.99ab 90.0a 97.4a 116a 0.45a 112a 116a 121b 0.12b Starch Pullulanase NGNS2 hr70.8ab 75.3a 89.3ab 3.96a 94.2a 103a 113a 0.92a  96.8a 124a 127ab 0.11bNGNS4 hr 69.7ab 74.8a 88.6ab 3.56a 69.2a  73.6a  79.4a 0.32a 117a 120a131ab 0.00835a NGNS16 70.3ab 75.3a 88.1ab 3.82a 93.6a 103a 111a 0.49a NDND ND ND hr GNS2 hr ND ND ND ND 90.2a 104a 115a 2.05a 118a 121a 129ab0.3b GNS4 hr ND ND ND ND 88.1a 105a 116a 2.17a 119a 122a 130ab 0.32bGNS16 hr ND ND ND ND ND ND ND ND 120a 122a 127ab 0.04b GS2 hr 73.1a81.3a 92.8ab 0.212c 97.0a 104a 112a 0.32a 119a 122a 126ab 0.05b GS4 hr62.8b 79.1a 96.7a 1.23bc 99.0a 105a 113a 0.32a 120a 122a 125ab 0.02bGS16 hr 63.9b 74.2a 85.5ab 0.397c 99.7a 112a 121a 179a    119a 125a 133a0.18b¹T_(o,) T_(p,) T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

Example 20

Effects of α-Amylase Treatment on the Heating Profile of Rice Starch

For α-amylase-treated rice starch, the gelatinization peak onset rangewas 56.3 to 76.6° C., peak range was 68.2 to 92.1° C., and completionrange was 86.4 to 106° C. (Table 18). No differences were found betweenany sample and the untreated rice starch in gelatinization temperaturesand enthalpies, except GS2hr had significantly greater peak andcompletion temperatures. The GNS4hr and 16 hr, and GS4hr-treated samplesdid not have a gelatinization peak. The enthalpy of peaks ranged from0.43 to 3.29 J/g, and the NGNS2hr-treated sample had the highestenthalpy, 4.03 J/g.

For the amylose-lipid complex, the peak onset range was 91.8 to 105° C.,peak range was 103 to 122° C., and completion range was 92.3 to 139° C.The GNS4hr-treated sample had the lowest onset and peak temperatures,and GS16hr sample had the highest. The non-significant differencesbetween the onset, peak and completion temperatures were due toinconsistent data from DSC analysis. Different treatments caused thepeak onset and peak temperatures to vary slightly (Table 18; heatprofile graphs not shown). No significant difference was seen in thecompletion temperatures and peak enthalpies. The peak enthalpies rangedfrom 0.23 to 4.45 J/g; the lowest was the GNS4hr-treated sample, and thehighest was the NGNS16hr sample.

The NGNS2hr, GNS2hr and 4 hr, and GS16hr-treated samples did not have aresistant starch peak. The GS4hr-treated sample had the lowesttemperature for peak onset (111° C.), peak (119° C.), and completion(126° C.). The highest temperature for peak onset was 124° C. inNGNS16hr sample, for peak was 125° C. in NGNS16hr sample, and forcompletion was 136.0° C. in GNS16hr sample. The non-significantdifferences between the wide temperature ranges were due to inconsistentdata from DSC analysis. The treatment with the highest enthalpy, 7.42J/g, was NGNS16hr, while GS4hr had the lowest enthalpy, 0.343 J/g. TABLE18 Effects of Gelatinization/Storage and Incubation Duration on ThermalCharacteristics of α-Amylase Treated Rice Starch^(1,2,3) FirstTransition Second Transition Third Transition (Gelatinization)(Amylose-lipid complex) (Resistant Starch) Sample Treatment T_(o) T_(p)T_(C) ΔH T_(o) T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH Control — ND ND ND NDND ND ND ND 101b 119a 133a 3.69a Rice — 68.8ab 73.3b 83.6b  2.99a  89.8a 97.4b 116a 0.450a 112ab 116a 121a 0.124a Starch α- NGNS2 hr 70.6ab75.7b 91.7ab 4.03a  96.3a 104ab 116a 0.360a ND ND ND ND Amylase NGNS4 hr74.9a 78.2b 90.7ab 3.29a 100a 104ab 111a 0.229a 116ab 119a 131a 1.04aNGNS16 hr 76.6a 79.8ab 92.6ab 2.68a 100a 106ab  92.3a 4.45a 124a 125a134a 7.42a GNS2 hr 56.3b 68.2b 86.6b  0.434a  95.9a 110ab 126a 0.39a NDND ND ND GNS4 hr ND ND ND ND 100a 111ab 122a 0.221a ND ND ND ND GNS16 hrND ND ND ND  91.8a 105ab 116a 0.669a 114ab 121a 136a 0.551a GS2 hr 76.7a92.1a 106a    2.14a  98.0a 103ab 113a 1.64a 113ab 120a 130a 0.3490 GS4hr ND ND ND ND ND ND ND ND 111ab 119a 126a 0.343a GS16 hr 64.0ab 73.3b86.4b  0.597a 105a 122a 139a 3.20a ND ND ND ND¹T_(o,) T_(p,) T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

Example 21

Effects of α-Amylase-Pullulanase Treatment on the Heating Profile ofRice Starch

For the α-amylase-pullulanase enzyme treatments, GNS4hr, GS2hr, 4 hr and16 hr did not have a gelatinization peak (Table 19; heating profilegraphs not shown). The NGNS4hr-treated sample had the highest enthalpyat 4.77 J/g, while the NGNS16hr-treated sample had the lowest, 0.146J/g. The enthalpies ranged from 0.146 to 4.77 J/g. The GNS2hr and 16hr-treated samples had a gelatinization peak during DSC analysis,probably indicating that the gelatinization prior to enzyme incubationwas incomplete. The peak onset range was 60.8 to 74.5° C., peak rangewas 72.9 to 78.1° C., and completion range was 81.8 to 96.5° C. (Table19) The NGNS16hr and GNS2hr-treated samples were significantly lowerthan the NGNS2hr and 4 hr treated samples and the untreated rice starchin peak onset temperature (p≦0.05). No significant difference was foundin the peak or peak completion temperature for NGNS, GNS2hr and 16 hrtreatments, and the untreated rice starch. The NGNS16hr treated samplehad a lower peak enthalpy than the NGNS4hr treated sample (p≦0.05).

For the GNS4hr-treated sample, no amylose-lipid complex peak wasdetected (Table 19). The remaining treatments, NGNS (all), GNS2hr and 16hr, and GS (all), were not significantly different from the untreatedrice starch for presence of amylose-lipid complex. The enthalpies forall the treated samples and untreated rice starch ranged from 0.177 to12.0 J/g, but were not significantly different from each other. The peakonset range was 84.3 to 105° C., peak range was 93.5 to 114° C., andcompletion range was 105 to 122° C.

The resistant starch peak temperatures for the α-amylase-pullulanasetreated rice starch were not significantly different from the commercialcontrol or from the untreated rice starch. The peak enthalpies rangedfrom 0.071 to 10.2 J/g. The lowest peak enthalpy was 0.071 J/g for theNGNS2hr sample (Table 19), and the highest for GNS16hr, 10.2 J/g. Thepeak onset range was 109 to 120° C., peak range was 115 to 126° C., andcompletion range was 122 to 137° C. TABLE 19 Effects ofGelatinization/Storage and Incubation Duration on ThermalCharacteristics of α-Amylase-Pullulanase Treated Rice Starch^(1,2,3)First Transition Second Transition Third Transition (Gelatinization)(Amylose-lipid complex) (Resistant Starch) Sample Treatment T_(o) T_(p)T_(C) ΔH T_(o) T_(p) T_(C) ΔH T_(o) T_(p) T_(C) ΔH Control — ND ND ND NDND ND ND ND 101a 119a 133a 3.69a Rice — 68.8bc 73.3a 83.6a 2.99ab 89.8a 97.4a 116a 0.45a 112a 116a 121a 0.124a Starch α- NGNS2 hr 72.5a 76.9a92.8a 3.47a 98.2a 102a 108a 0.178a  83.8a  84.7a  87.8a 0.071a Amylase-NGNS4 hr 74.5a 77.3a 90.8a 4.77a 97.0a 100a 106a 2.22a 114a 116a 122a0.808a Pullulanase NGNS16 hr 62.6d 72.9a 81.8a 0.146b 101a   103a 113a0.177a 120a 122a 130a 0.095a GNS2 hr 60.8d 78.1a 96.5a 1.92ab 105a  114a 122a 0.143a ND ND ND ND GNS4 hr ND ND ND ND ND ND ND ND 114a 126a137a 0.31a GNS16 hr 63.7cd 74.5a 85.2a 0.727b 98.2a 106a 113a 0.189a117a 123a 131a 0.369a GS2 hr ND ND ND ND 84.3a  93.5a 105a 1.09a 117a123a 131a 0.37a GS4 hr ND ND ND ND 95.1a  98.5a 112a 12.0a 114a 122a133a 0.999a GS16 hr ND ND ND ND 88.6a  97.7a 108a 6.38a 109a 115a 125a10.2a¹T_(o,) T_(p,) T_(C) = onset, peak and completion temperatures,respectively; ΔH = enthalpy; ND = non-detectable.²Means with different letters within each column are significantlydifferent at p ≦ 0.05. The values are an average of 2 to 4 measures.³Units: Temperature (° C.), Enthalpy (J/g, dry matter); Heating Rate =5° C./min

Example 22

Stability of Resistant Starch Peaks

When a resistant starch peak was identified in DSC from the treated riceflour and starch samples, the same sample was reheated to 140° C. toexamine the heat stability of resistant starch. The 27 samples found tohave resistant starch that was heat stable are presented in Table 20.

Five rice flour treatments increased in peak enthalpy during reheating:three pullulanase samples, GNS2hr, GS2hr and 4 hr; and twoα-amylase-pullulanase samples, NGNS2hr and GS4hr. In the rice starchsamples, GS2hr (pullulanase) was the only sample that indicated anincrease in peak enthalpy during reheating. The peak enthalpies of GS16h(pullulanase, rice starch) and GS4hr and 16 hr (α-amylase-pullulanase,rice starch) were significantly reduced to about 3% after reheating. Noclear pattern was detected on the influence of gelatinization,incubation time, and type of enzyme on the heat stability of resistantstarch.

For both rice flour and starch, all three enzyme treatments producedresistant starch according to the DSC analysis. Pullulanase treatmentsproduced the most samples that had heat stable resistant starch,followed by α-amylase-pullulanase, and then α-amylase. This trend wasobserved in both rice flour and starch.

The resistant starch formed from pullulanase treated starch consisted ofboth linear amylose chains cleaved from amylopectin and original amylosechains. The resistant starch present in the α-amylase andα-amylase-pullulanase treatments, however, probably had fewer andshorter linear amylose chains due to the random cleaving by α-amylase.When pullulanase debranched the starch molecules in theα-amylase-pullulanase treatment, the linear chains became highlyaccessible to the α-amylase, and greater amount of degradation to thestarch molecules occurred. TABLE 20 Enthalpy of Heat-stable ResistantStarch Peaks Original Sample Treatment ΔH (J/g) Final H (J/g) %Remaining RiceStarch Pullulanase NGNS2 hr 2.11 0.745 35.3 NGNS4 hr 0.7780.764 98.2 GNS2 hr 2.73 2.05 75.1 GSN4 hr 2.48 2.26 91.1 GNS16 hr 8.815.6 63.6 GS2 hr 0.349 0.969 278 GS16 hr 569 12.0 2.1 Amylase- NGNS4 hr8.88 1.36 15.3 pullulanase GS2 hr 2.86 0.62 21.7 GS4 hr 65.19 2.17 3.3GS16 hr 11.51 0.256 2.2 Amylase NGNS4 hr 3.84 2.11 54.9 GS2 hr 1.79 0.5631.3 GS16 hr 1.65 1.6 97.0 Flour Pullulanase NGNS2 hr 4.73 2.7 57.1NGNS4 hr 4.31 0.893 20.7 GNS2 hr 0.971 1.18 122 GNS16 hr 8.17 5.05 61.8GS2 hr 0.406 1.78 438 GS4 hr 0.526 1.67 317 GS16 hr 3.89 2.42 62.1Amylase- NGNS2 hr 0.225 0.329 146 pullulanase GS2 hr 351 72.9 20.8 GS4hr 3.35 8.06 241 GS16 hr 152 49.9 32.8 Amylase GS2 hr 373 69.9 18.7 GS4hr 213 127 59.6

Of all the non-gelatinized (NGNS) treatments on rice flour and ricestarch, only three samples did not have a gelatinization peak: NGNS16hr(α-amylase-pullulanase, rice flour), and NGNS2hr and 4 hr (α-amylase,rice flour). Although some of the GNS and GS samples had gelatinizationpeaks when analyzed with DSC, this was probably due to incompletegelatinization during sample preparation

Only three rice starch samples did not have a second transition peak:GNS16hr (pullulanase), GNS4hr (α-amylase-pullulanase), and GS4hr(α-amylase). The highest peak enthalpy was 179 J/g in a single GS16hrtreated sample (pullulanase, rice starch). The rest of the treatedsamples (rice flour and starch) had enthalpies between 0.5 and 2 J/g.

Resistant starch was detected in most of the enzyme treated samples. Theenzyme combinations, incubation time, and gelatinization type did notcause a significant difference in the thermal properties of theresistant starch formed from rice flour and rice starch. The resistantstarch peak was heat stable. Heat stability is important because thenresistant rice starch can be incorporated into a wider variety of foodproducts, such as frozen dinners that require a second heating beforeconsumption.

Enzyme treatments on rice starch and flour produced resistant starch(RS) yields on a wide scale. The RS yields were dependent on the sourceof starch, gelatinization type, incubation period, and enzymecombination. The rice starch produced a higher RS yield than rice flourfor most of the treatments. The non-gelatinized (NGNS) treatmentsyielded the highest RS content for both rice starch and rice flour. Inrice starch, the NGNS (pullulanase) treated samples had a yield of 40 to61% RS (based on dry weight). There was no discernable trend inincubation time. Some 16 hr treatments had significantly higher yieldswhile others had lower yields. For enzymatic treatments, pullulanaseproduced the best resistant starch yields in both rice starch and flour.The lower yields seen in α-amylase-pullulanase and α-amylase treatmentswere attributed to the random cleaving effects of α-amylase, degradingthe amylose present.

Gelatinization, regardless of enzyme treatment, deteriorated orminimized the pasting characteristics of the rice flour and starch, asanalyzed using rapid visco amylograph (RVA). Thenon-gelatinized-no-overnight-storage (pullulanase) samples had the bestpasting characteristics among all the treatments, probably due to themilder temperature (55° C.) of incubation and the pattern of enzymecleavage on starch. The non-gelatinized, pullulanase samples were alsothe most similar in pasting characteristics to the untreated rice flourand starch.

DSC analysis of the samples was difficult to interpret due to largevariations in the data, and because some of the pre-gelatinized samplesindicated the presence of gelatinization peaks, probably indicating anincomplete initial gelatinization process. Amylose-lipid complex andresistant starch were detected in both rice starch and rice floursamples. The samples with resistant starch peaks were reheated to testthe heat stability of the resistant starch present. A number of samplesfrom both rice flour and rice starch tested positive for resistantstarch heat stability. Three samples (GS16hr (pullulanase, rice starch),and GS4hr and 16 hr (α-amylase-pullulanase, rice starch)) retained aresistant starch peak during reheating; however the peak enthalpies werereduced to about 3% of the initial peak enthalpy.

Non-gelatinized rice starch treated with pullulanase for 2 to 4 hoursyielded the highest amounts of resistant starch levels that retained itspasting characteristics. This resistant rice starch may be used as avalue-added food ingredient. CrystaLean®, the commercial resistantstarch made from corn, is currently used in diabetic candy bars as abulking agent. The rice resistant starch as produced by pullulanasetreatment on non-gelatinized samples may have wider range of use as therice resistant starch retained its pasting properties while CrystaLean®did not. This specific treatment produced a starch with same pastingcharacteristics as untreated rice starch, but with 8 to 12 times moreresistant starch (fiber). The resistant starch was also heat resistant,as a peak was detected during reheating. This was significant becausethis resistant rice starch could be used in food products that areheated, and have a high viscosity. It could also be incorporated intofrozen dinners where reheating is a prerequisite. Rice is alsohypoallergenic due to its low protein content, and would therefore beless likely to cause food allergies in consumers. Moreover, resistantstarch was formed from starch with less than 30% amylose and withoutheating the starch above 60° C.

As used in the specification and the claims, the term “native starch”means a starch that has not been pre-treated, including starch that hasnot been heated to cause gelatinization or treated chemically orenzymatically to cause hydrolysis.

The complete disclosures of all references cited in this application arehereby incorporated by reference. Also, incorporated by reference is thecomplete disclosure of the following documents: Siow Ying Tan,“Resistant Rice Starch Development,” A thesis submitted to theDepartment of Food Science, Louisiana State University, August, 2003; S.Y. Tan and J. M. King, “Enzymatic treatment to form resistant ricestarch,” An abstract for the 2003 Annual Meeting of the Institute ofFood Technologists, published online March 2003; and S. Y. Tan and J. M.King, “Enzymatic Treatment to form Resistant Rice Starch,” a posterpresented on Jul. 14, 2003, at the 2003 Annual Meeting of the Instituteof Food Technologists, Chicago, Ill. In the event of an otherwiseirreconcilable conflict, however, the present specification shallcontrol.

1. A resistant starch product produced from a native starch, wherebysaid product exhibits a pasting temperature and a peak viscosity that iswithin 25% of that exhibited by the native starch, contains feweralpha-1,6-glucosidic bonds than the native starch, and contains a higherpercentage of starch molecules that are resistant to alpha-amylasedigestion.
 2. A resistant starch product as in claim 1, wherein saidnative starch is selected from the group consisting of rice starch,flour starch, potato starch, corn starch, wheat starch, barley starch,tapioca starch, cassava starch, arrowroot starch, sago starch, and oatstarch.
 3. A resistant starch product as in claim 2, wherein said nativestarch is rice starch.
 4. A resistant starch product as in claim 1,wherein said native starch contains less than 30% amylose.
 5. Aresistant starch product as in claim 1, wherein said product has fromabout a three-fold to about a twelve-fold increase in the percentage ofstarch molecules that are resistant to alpha-amylase digestion ascompared to the native starch.
 6. A food product comprising a resistantstarch product as in claim
 1. 7. A method to produce a resistant starchproduct by digestion of a native starch, said method comprisingpreparing an aqueous slurry of the native starch, incubating the slurrywith an effective amount of a debranching enzyme to hydrolyze1,6-glucosidic bonds of starch molecules at a temperature less than 60°C., and isolating the resistant starch product.
 8. A method as in claim7, wherein said starch is selected from the group consisting of ricestarch, flour starch, corn starch, potato starch, wheat starch, barleystarch, tapioca starch, cassava starch, arrowroot starch, sago starch,and oat starch.
 9. A method as in claim 8, wherein the starch is ricestarch.
 10. A method as in claim 7, wherein the starch contains lessthan 30% amylose.
 11. A method as in claim 7, wherein the debranchingenzyme is selected from the group consisting of pullulanase andisoamylase.
 12. A method as in claim 7, wherein the debranching enzymeis pullulanase.
 13. A method as in claim 7, wherein the incubationtemperature is from about 45° C. to about 60° C.
 14. A method as inclaim 7, wherein the incubation temperature is about 55°.
 15. A methodas in claim 7, wherein the incubation is for about 2 hours to about 16hours.
 16. A method as in claim 15, wherein the incubation is for about2 hours to about 4 hours.
 17. A method as in claim 15, wherein theincubation is for about 4 hours.
 18. A method as in claim 7, wherein theresistant starch product exhibits a pasting temperature and a peakviscosity that is within 25% of that exhibited by the native starch. 19.A method as in claim 7, wherein said resistant starch product containsfrom about a three-fold to about a twelve-fold increase in thepercentage starch molecules that are resistant to alpha-amylasedigestion as compared to the native starch.